HMG2 promoter expression system and post-harvest production of gene products in plants and plant cell cultures

ABSTRACT

The invention relates in part to plant HMG2 HMGR genes and in part to the “post-harvest” production method of producing gene product of interest in plant tissues and cultures. The HMG2 promoter elements are responsive to pathogen-infection, pest-infestation, wounding, or elicitor or chemical treatments. The HMG2 elements are also active in specialized tissues of the plant including pollen and mature fruits. HMG2 promoter elements and HMG2-derived promoters can be advantageously used to drive the expression of disease and pest resistance genes, whereby transgenic plants containing such gene constructs would be resistant to the targeted disease and pest. In particular, the HMG2 gene expression system can be utilized in developing nematode resistant plants. The post-harvest production method of the invention utilizes plant tissues and cell cultures of plants or plant cells engineered with a expression construct comprising an inducible promoter, such as the HMG2 promoter, operably linked to a gene of interest. Production of the desired gene product is obtained by harvesting, followed by inducing and processing the harvested tissue or culture. The post-harvest production method may be advantageously used to produce direct or indirect gene products that are labile, volatile, toxic, hazardous, etc.

[0001] This application is a continuation-in-part of co-pendingapplication Ser. No. 08/100,816 filed Aug. 2, 1993, which is herebyincorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates in part to HMG2 promoters, activefragments thereof, and their use to drive the expression of heterologousgenes. Expression vectors may be constructed by ligating a DNA sequenceencoding a heterologous gene to an HMG2 promoter or a promoter modifiedwith an HMG2 cis-regulatory element. Such constructs can be used for theexpression of proteins and RNAs in plant cell expression systems inresponse to elicitor or chemical induction, or in plants in response towounding, pathogen infection, pest infestation as well as elicitor orchemical induction.

[0003] The present invention also relates to a method of producing geneproducts in harvested plant tissues and cultures. The “post-harvest”production method utilizes tissues or cultures from transgenic plants orplant cells, respectively, engineered with expression constructscomprising inducible promoters operably linked to sequences encodingdesirable protein and RNA products. Treatment of the harvested tissuesor cultures with the appropriate inducer and/or inducing conditionsinitiates high level expression of the desired gene product. Thepost-harvest production method may advantageously be used to producegene products and metabolites of gene products that are labile, volatileor toxic or that are deleterious to plant growth or development.

[0004] This aspect of the invention is demonstrated by way of exampleswhich describe the production of transgenic plants engineered withexpression constructs comprising inducible promoters operably linked tocoding sequences of interest, and which show that days-old and weeks-oldharvested tissues from such transgenic plants remain capable of inducedproduction of high levels of the gene product of interest.

2. BACKGROUND OF THE INVENTION

[0005] Plant diseases caused by fungal, bacterial, viral and nematodepathogens cause significant damage and crop loss in the United Statesand throughout the world. Current approaches to disease control includebreeding for disease resistance, application of chemical pesticides, anddisease management practices such as crop rotations. Recent advances inplant biotechnology have led to the development of transgenic plantsengineered for enhanced resistance to plant pests and pathogens. Thesesuccesses indicate that biotechnology may significantly impactproduction agriculture in the future, leading to improved cropsdisplaying genetic resistance. This mechanism of enhanced diseaseresistance has the potential to lower production costs and positivelyimpact the environment by reducing the use of agrichemicals.

[0006] Recent advances in plant biotechnology have led also to thedevelopment of transgenic plants engineered for expression of newfunctions and traits. Such engineering has produced transgenic plantshaving new synthetic capabilities. These successes indicate thatbiotechnology may enhance the role of plants as a producer of chemicals,pharmaceuticals, polymers, enzymes, etc. The engineering of novelsynthetic capabilities has the potential to increase crop values as wellas create new uses for crops such as tobacco.

2.1. Plant Defense Responses

[0007] Higher plants have evolved a variety of structural and chemicalweapons for protection against pathogens, predators, and environmentalstresses. Expression of resistance toward pathogens and pests involvesthe rapid induction of genes leading to accumulation of specificdefense-related compounds (Dixon et al., 1990, Adv. Genet. 28:165-234).These responses include accumulation of phytoalexin antibiotics,deposition of lignin-like material, accumulation of hydroxyproline-richglycoproteins, and increases in hydrolytic enzymes such as chitinase andglucanase.

[0008] Phytoalexins are low molecular weight compounds which accumulateas a result of pathogen challenge. Phytoalexins have antimicrobialactivity and have proved to be of critical importance in diseaseresistance in several plant:pathogen interactions (Darvill et al., 1984,Annu. Rev. Plant Physiol. 35:243; Dixon et al., 1990, Adv. Genet.28:165-234; and Kuc and Rush, 1895, Arch. Biochem. Biophys.236:455-472). Terpenoid phytoalexins represent one of the major classesof plant defense compounds and are present across a wide range of plantspecies including conifers, monocots and dicots. The regulation ofterpenoid phytoalexin biosynthesis has been most extensively studiedamong the solanaceae (e.g., tomato, tobacco, potato), however, mostimportant crop species utilize this pathway in disease resistance.

[0009] In well characterized plant:microbe interactions, resistance topathogens depends on the rate at which defense responses are activated(Dixon and Harrison, 1990, Adv. Genet. 28:165-234). In an incompatibleinteraction, the pathogen triggers a very rapid response, termedhypersensitive resistance (HR), localized to the site of ingress. In thesusceptible host, no induction of defense responses is,seen untilsignificant damage is done to host tissue.

[0010] Defense-responses, including phytoalexin accumulation, can alsobe induced in plant cell cultures by compounds, termed “elicitors”, forexample components derived from the cell walls of plant pathogens(Cramer et al., 1985, EMBO J. 4:285-289; Cramer et al., 1985, Science227:1240-1243; Dron et al., 1988, Proc. Natl. Acad. Sci. USA85:6738-6742). In tobacco cell cultures, fungal elicitors trigger acoordinate increase in sesquiterpene phytoalexin biosynthetic enzymesand a concomitant decrease in squalene synthetase which directs isopreneintermediates into sterol biosynthesis (Chappell and Nable, 1987, PlantPhysiol. 85:469-473; Chappell et al., 1991, Plant Physiol. 97:693-698;Threlfall and Whitehead, 1988, Phytochem. 27:2567-2580).

2.2. Physiological Roles of HMGR in Plants

[0011] A key enzyme involved in phytoalexin biosynthesis and, hence,plant defense against diseases and pests, is 3-hydroxy-3-methylglutarylCoA reductase (HMGR; EC 1.1.134). HMGR catalyzes the conversion of3-hydroxy-3-methylglutaryl CoA to mevalonic acid. Mevalonic acid is anintermediate in the biosynthesis of a wide variety of isoprenoidsincluding defense compounds such as phytoalexin.

[0012] In addition to phytoalexin biosynthesis, themevalonate/isoprenoid pathway produces a diverse array of otherbiologically important compounds (see FIG. 1). These include growthregulators, pigments, defense compounds, UV-protectants, phytotoxins,and specialized terpenoids such as taxol, rubber, and compoundsassociated with insect attraction, fragrance, flavor, feeding deterrenceand allelopathy. Many of these compounds have critical roles in cellularand biological functions such as membrane biogenesis, electrontransport, protein prenylation, steroid hormone synthesis, intercellularsignal transduction, photosynthesis, and reproduction.

[0013] The conversion of 3-hydroxy-3-methylglutaryl CoA to mevalonicacid appears to be the rate-limiting step of isoprenoid biosynthesis.Thus, the regulation of HMGR expression serves as a major control pointfor a diversity of plant processes including defense responses. PlantHMGR levels change in response to a variety of external stimuliincluding light, plant growth regulators, wounding, pathogen attack, andexogenous sterols (Brooker and Russell, 1979, Arch. Biochem. Biophys.198:323-334; Russell and Davidson, 1982, Biochem. Biophys. Res. Comm.104:1537-1543; Wong et al., 1982, Arch. Biochem. Biophys. 216:631-638and Yang et al., 1989, Mol. Plant-Microb. Interact. 2:195-201) and varydramatically between tissues and developmental stages of plant growth(Bach et al., 1991, Lipids 26:637-648 and Narita and Gruissem, 1991, J.Cell. Biochem 15A:102 (Abst)). Reflecting this complexity, multiple HMGRisozymes exist in plants which are differentially regulated and may betargeted to distinct organellar locations. In addition to microsomallocations, plant HMGR isozymes have been localized to mitochondrial andchloroplast membranes (Brooker and Russell, 1975, Arch. Biochem.Biophys. 167:730-737 and Wong et al., 1982, Arch. Biochem. Biophys.216:631-638).

2.3. Plant HMGR Genes

[0014] HMGR genomic or cDNA sequences have been reported for severalplant species including tomato (Narita and Gruissem, 1989, Plant Cell1:181-190; Park, 1990, Lycopersicon esculentum Mill. Ph.D. Dissertation,Virginia Polytechnic Institute and State University, Blacksburg, Va.),potato (Choi et al., 1992, Plant Cell 4:1333-1344 and Stermer et al.,1991, Physiol. Mol. Plant Pathol. 39:135-145), radish (Bach et al.,1991, American Oil Chemists Society, Champaign, Ill. page 29-49),Arabidopsis thaliana (Caelles et al., 1989, Plant Mol. Biol. 13:627-638;Learned and Fink, 1989, Proc. Natl. Acad. Sci. USA 86:2779-2783),Nicotiana sylvestris (Genschik et al., 1992, Plant Mol. Biol.20:337-341), Catharanthus roseus (Maldonado-Mendoza et al., 1992, PlantPhysiol. 100:1613-1614), the Hevea rubber tree (Chye et al., 1991, PlantMol. Biol. 16:567-577) and wheat (Aoyagi et al., 1993, Plant Physiol.102:622-638). Cloning of additional HMGR genes from rice (Nelson et al.,1991, Abstract 1322, 3rd Intl. Cong. Intl. Soc. Plant Mol. Biol.,Tucson, Ariz.), and tomato (Narita et al., 1991, J. Cell. Biochem15A:102 (Abst)) also has been reported but no DNA sequence informationhas been published for these HMGR genes. In all plants thus farinvestigated, HMGR is encoded by a small gene family of two(Arabidopsis) or more members (tomato, potato, Hevea). See Table 1 for asummary of published information pertaining to plant HMGR genes. TABLE 1+UZ,1/55 SUMMARY OF CLONED PLANT HMGR SEQUENCES AND THEIR REGULATION.(Footnotes and references on next page) HMGR Regulation Promoter^(c)Species gene Description^(a) constit. pollen wound pathogen^(b) otherAnalysis Ref. Lycopersicon hmg2 G, cDNA, F, S − + + B, F, E, N maturefruit S, GUS, D 1 esculentum hmg1 cDNA, P, S low ? ? — immat. fruit — 1,2 (tomato) Solanum hmg1 cDNA, F, S low ? + — elic. suppressed GUS 3, 4tuberosum hmg2 cDNA, P, S − ? + B, E — — 3 (potato) hmg3 cDNA, P, S −? + B, E — — 3 Nicotiana hmgr cDNA, F, S + ? E, V roots, protoplasts — 5sylvestris Arabidopsis hmg1 cDNA, G, F, S low ? ? ? — — 6 thaliana hmg2cDNA − ? ? ? — — 7 Hevea hmg1 G, cDNA, F, S − ? ? ? laticifer, ethyleneind — 8 brasiliensis hmg2 cDNA, P, S ? ? ? ? — — 9 hmg3 cDNA, F, S +“housekeeping” S 8 Catharanthus hmgr cDNA — — 10 Raphanus sativus hmg1cDNA, F, S ? ? ? ? — — 11 (radish) hmg2 cDNA, F, S ? ? ? ? isolatedseedlings — Triticum hmgr10 cDNA, P low ? − ? callus — 12 aestivumhmgr18 cDNA, P + ? − ? callus — (wheat) hmgr23 cDNA, P − ? − ? roots,callus —

2.4. Tomato HMGR Genes

[0015] Tomato HMGR genes have been isolated by direct probing of atomato genomic library with yeast HMG1 cDNA sequences (Cramer et al.,1989, J. Cell. Biochem. 13D:M408; Park, 1990, Lycopersicon esculentumMill. Ph.D. Dissertation, Virginia Polytechnic Institution and StateUniversity, Blacksburg, Va.). One of these genes, designated tomatoHMG2, has been characterized further and the sequence of the codingregion reported (Park et al., 1992, Plant Mol. Biol. 20:327-331; GenBankM63642). The nucleic acid sequence of tomato HMG2 has been compared tothose of other plant HMGRs. Sequence comparisons within regions encodingthe HMGR protein show identities in the range of 69 to 96%, with themost closely related sequences being a partial cDNA sequence of potatoHMG2 (96% sequence identity for 744 base at the C-terminus codingregion) and an HMGR cDNA from Nicotiana sylvestris (89% sequenceidentity; entire coding region). The tomato HMG2 gene is distinct fromtomato HMG1 showing 78% sequence identity within the region encoding theN-terminus (1067 bases compared; see FIG. 2). Comparisons of sequencesoutside the coding region (e.g., the 5′-untranslated leader sequence)reveal very little sequence conservation between divergent species andonly limited homology among HMGR genes within the Solanaceae. TomatoHMG2 shows less than 75% sequence identity over an 81 base stretch withthe N. Sylvestris leader sequences. Comparisons between the 5′-leadersequences of tomato HMG1 and HMG2 are included in FIG. 2.

[0016] The coding region of the tomato HMG2 gene is interrupted by threeintrons and encodes a protein with a calculated molecular mass of 64,714(Park et al., 1992, Plant Mol. Biol. 20:327-331). Table 2 shows acomparison of the derived amino acid sequence of the tomato HMG2 toHMGRs of other organisms. The N-terminal membrane domain of the tomatoHMG2 HMGR is significantly smaller, shows no sequence similarity to theyeast or animal transmembrane domain, and contains 1-2 potentialmembrane spanning regions compared to 7-8 postulated in non-plant HMGRs(Basson et al., 1988, Mol. Cell Biol. 8:3797-3808; Liscum et al., 1985,J. Biol. Chem 260:522-530). This transmembrane domain is also highlydivergent among plant species and among HMGR isogenes within a speciesalthough the actual membrane spanning residues are quite conserved.

[0017] Amino acid sequence comparisons between the N-terminal twohundred residues of tomato HMG2 HMGR and other plant HMGRs yieldsequence identities in the range of 44-85% (Table 2). Tomato HMG1 andHMG2 show only 75% sequence identity in this region. The catalyticdomain of tomato HMG2, located within the C-terminal 400 amino acids,shows significant sequence homology with the yeast (56% identity; 75%similarity) and human (55% identity; 75% similarity) HMGRs. This domainis highly conserved between plant species: comparisons between tomatoHMG2 HMGR (residues 200-602) and other plant HMGRs yield amino acididentities ranging from 74-90% (see Table 2).

[0018] In contrast to the information available on plant HMGR codingsequences, relatively little is known of the structure of plant HMGRpromoters. Indeed, to date, only one plant HMGR promoter sequence hasbeen reported (Chye et al., 1991, Plant Mol. Biol. 16:567-577). TABLE 2Amino acid identity (%) of the membrane and catalytic domains of plantHMGRs. Solanaceae Euphorb. Apo Brassicaceae Tom1 Tom2 Pot1 Pot3 Nic Hev1Hev3 Car Ara1 Rad1 Rad2 Tom1 100 75 98 — 77 46 53 64 57 48 57 — — — — —— — — — — — Tom2 100 74 — 85 44 52 66 50 48 52 100 90 90 95 85 74 84 8180 80 Pot1 100 — 76 45 54 63 58 49 57 100 90 93 88 85 88 83 83 83 Pot3 —— — — — — — — 100 92 86 85 84 82 81 82 Nic 100 42 49 63 54 49 57 100 8786 87 83 83 82 Hev1 100 44 38 76 51 55 100 86 87 86 86 85 Hev3 100 46 5648 48 100 85 82 82 82 Car 100 48 44 49 100 81 82 82 Ara1 100 73 78 10094 95 Rad1 100 72 100 93 Rad2 100 100 # roseus (Car, isogene unknown),and R. sativus (radish; Rad1, Rad2). Comparisons are based on thesequences available from GenBank.

2.5 The Functions and Expression Patterns of Different HMGR Genes

[0019] Specific plant HMGR isogenes show very distinct patterns ofregulation during development and in response to pathogen infection orpest infestation (Caelles et al., 1989, Plant Mol. Biol. 13:627-638;Choi et al., 1992, Plant Cell 4:1333-1344; Chye et al., 1992, Plant Mol.Biol. 19:473-484; Narita and Gruissem, 1989, Plant Cell 1:181-190; Parket al., 1992, Plant Mol. Biol. 20:327-331; Park, 1990, Lycopersiconesculentum Mill. Ph.D. Dissertation, Virginia Polytechnic Institute andState University, Blacksburg, Va.; Yang et al., 1991, Plant Cell3:397-405).

[0020] In the Solanaceae, the pathogen-induced synthesis of phytoalexinantibiotics is due to the induction of microsome-associated HMGR (Shihand Kuc, 1973, Phytopathology 63:826-829 and Stermer and Bostock, 1987,Plant Physiol. 84:404-408). In cultured cells, the induction occurs withthe addition of elicitors isolated from the cell walls of pathogenicmicrobes (Chappell et al., 1991, Plant Physiol. 97:693-698 and Stermerand Bostock, 1987, Plant Physiol. 84:404-408). Typically, elicitortreatment results in a marked, transient increase in HMGR mRNA levelthat is typical of transcriptionally regulated defense genes in plants(Cramer et al., 1985, EMBO J. 4:285-289; Dixon and Harrison, 1990, Adv.Genet. 28:165-234; Yang et al., 1991, Plant Cell 3:397-405).

[0021] The rapid pathogen-induced HMGR expression appears to be encodedby the HMG2 HMGR isogene. This has been established in tomato cellstreated with elicitors (Park, 1990, Lycopersicon esculentum Mill. Ph.D.Dissertation, Virginia Polytechnic Institute and State University,Blacksburg, Va.) and in potato tubers infected with the soft-rottingbacterium Erwinia carotovora (Yang et al., 1991, Plant Cell 3:397-405).In potato tuber, HMG2 mRNA levels increased 20-fold within 14 hrfollowing bacteria inoculation but was not induced by wounding in theabsence of pathogen (Yang et al., 1991, Plant Cell 3:397-405).

[0022] HMG2 expression has been shown to be critical to diseaseresistance. An elicitor, arachidonic acid, induces defense responsesincluding increases in HMG2 mRNA and thus, phytoalexin synthesis inpotato tubers (Stermer and Bostock, 1987, Plant Physiol. 84:404-408 andYang et al., 1991, Plant Cell 3:397-405). Tubers treated witharachidonic acid 48 hr prior to inoculation with Erwinia carotovora werecompletely resistant to rotting. Tubers inhibited in HMGR activity bymevinolin (an HMGR-specific competitive inhibitor) showed significantlyincreased rotting (Yang et al., 1991, Plant Cell 3:397-405).

[0023] Homologs to the tomato HMG2 HMGR isogene, based on analogousexpression patterns, exist in other plants. For example, potato HMG2 andHMG3 mRNAs also are induced in response to wounding, elicitors andbacterial pathogens (Choi et al., 1992, Plant Cell 4:1333-1344 and Yanget al., 1991, Plant Cell 3:397-405). An HMGR isogene isolated fromNicotiana sylvestris is induced by virus inoculation and defenseelicitors (Genschik et al., 1992, Plant Mol. Biol. 20:337-341).Similarly, an HMGR isogene of rice also is induced by wounding andelicitors (Nelson et al., 1991, Abst 1322, 3rd Intl. Cong. Intl. Soc.Plant Mol. Biol., Tucson, Ariz.) suggesting that defense-specific HMG2homologs are also present in monocotyledonous plants. However, the threeHMGR cDNAs isolated from wheat apparently are not wound-inducible(Aoyagi et al., 1993, Plant Physiol. 102:623-628). Although DNAsequences have not been isolated, increases in HMGR enzyme activity inresponse to bacterial or fungal pathogens have been documented in anumber of other plant species suggesting that the HMG2 isogene is widelyrepresented among plants.

[0024] Tomato HMG2 is quite distinct from the tomato HMG1 isogene at thenucleic acid level (FIG. 2). The HMG1 HMGR isogene appears to beexpressed under circumstances that are different from those that inducethe expression of the HMG2 gene. Narita and Gruissem have shown thattomato HMG1 is expressed at low levels in all tomato tissues analyzedand is highly expressed in immature fruit during the period of rapidgrowth (Narita and Gruissem, 1989, Plant Cell 1:181-190; Narita et al.,1991, J. Cell. Biochem. 15A:102 (Abst)). Thus, tomato HMG1 may functionin sterol synthesis and membrane biogenesis. Choi et al. have shown thatpotato HMG1 expression is induced in potato tubers by wounding but issuppressed by defense-elicitors or bacterial pathogens (Plant Cell4:1333-1344). These data suggest that the HMGR encoded by the HMG1 isassociated with the biosynthetic branch responsible for sterolproduction. Based on similarities in sequence or expression patterns(e.g., constitutive low level expression, suppression by elicitors ormicrobes, high expression during rapid cell division), Hevea HMGR3,potato HMG1, Arabidopsis HMG1, and radish HMG2 probably belong to thisHMG1 HMGR isogene class.

2.6. Plant HMGR Promoters

[0025] Little is known of the promoters of plant HMGR genes. To date,there have been no known publications of studies analyzing the structureof plant HMGR promoters (e.g., deletion analyses, DNaseI footprintanalysis, DNA-protein binding/gel retardation analyses). One article(Chye et al., 1991, Plant Mol. Biol. 16:567-577) documents approximately1.5 kb of the upstream sequence of the Hevea HMG1 gene. This sequenceshows no significant homology to the analogous region of the tomato HMG2promoter disclosed herein. Another report cursorily mentioned expressionof potato HMGR13 (presumably an HMG1 homolog) promoter:β-glucuronidase(GUS) fusions in transgenic tobacco (Stermer et al., 1992,Phytopathology 82:1085), but revealed no details concerning the promoterused nor the construction of the fusion.

2.7. Production of Desirable Gene Products in Plants

[0026] The concept of using transgenic plants to produce desirable geneproducts is by no means new. Virtually all examples reported to date ofsuch engineering of transgenic plants have utilized “constitutive”promoters, especially the cauliflower mosaic virus (CaMV) 35S promoteror enhanced versions thereof, to drive production of transgenicproducts. The following is a representative list of the many mammaliantherapeutic proteins that have been produced in plants using the CaMV35S promoter or derivatives. Expression of mouse immunoglobulin chainswas achieved by transforming tobacco leaf segments with gamma- orkappa-chain cDNAs derived from a mouse hybridoma cell line (Hiatt etal., 1989, Nature 342:76-78; Hein et al., 1991, Biotechnol. Prog.7:455-461). Transformed plants expressing either gamma or kappa chainswere genetically crossed resulting in the production of assembled,functional antibodies (termed “plantibodies”). Transgenic tobaccoproducing human serum albumin (Sijmons et al., 1990, Bio/Technology8:217-221), rabbit liver cytochrome P450 (Saito et al., 1991, Proc.Natl. Acad. Sci. USA 88:7041-7045), hamster 3-hydroxy-3-methylglutarylCoA reductase (Chappell et al., 1991, Plant Physiol. 96:127), and thehepatitis B surface antigen (Mason et al., 1992, Proc. Natl. Acad. Sci.USA 89:11745-11749) have also been reported. HSA produced as a fusionprotein with plant prepro-signals resulted in a secreted HSA proteinthat was indistinguishable from authentic mature human protein (Sijmonset al., 1990, Bio/Technology 8:217-221). The hepatitis Bantigen-expressing plants accumulated spherical HBsAg particles withphysical and antigenic properties similar to human serum-derived HBsAgparticles and to those generated in yeast (current source of humanvaccine). Plant virus-mediated expression of human proteins in plants,including human interferon, α- and β-hemoglobin, and melanin has alsobeen demonstrated (Fraley, R., 1992, Bio/Technology 10:36-43; de Zoetenet al., 1989, Virology 172:213-222).

[0027] The constitutive expression of desirable gene products, however,is disadvantaged whenever the desirable gene product is 1) labile, 2)deleterious to the growth or development of the transgenic plant orculture line, 3) toxic to humans, livestock, animals, or insects thatcould inadvertently eat the transgenic plant, 4) a real or potentialenvironmental hazard, or 5) a security risk due to the extremely highvalue of the gene product. Moreover, some constitutive promoters, suchas the CaMV 35S, decline in activity as plants matures. Such losses ofactivity can result in inefficient exploitation of the host plant as aproduction system, since mature or maturing plants have greater biomassand, often, greater excess biosynthetic capacity for the production ofthe “extraneous” desirable gene product than younger plants which areprogrammed to devote much of their biosynthetic capacities to normalgrowth and development functions.

[0028] Compared to the aforementioned conventional approach, thepost-harvest production method of the instant invention can be a muchmore efficient and productive means of producing desirable gene productsin plants. Using the post-harvest production method, the desired geneproduct is not produced until after the plant tissue or cell culture hasbeen harvested and induced. This obviates any problems that might arisefrom the presence of the desired gene product during the growth of theplant or culture line.

3. SUMMARY OF THE INVENTION

[0029] One aspect of the present invention relates to the use of plantpromoter sequences responsive to wounding, pathogen infection, pestinfestation as well as to elicitors or chemical inducers. This aspect ofthe invention generally relates to the use of promoter sequences of3-hydroxymethyl-3-glutaryl coA reductase (HMGR) genes, and specificallyto the tomato HMG2 (HMGR2) promoter element and its homologs from otherplant species, to control the expression of protein and RNA products inplants and plant cells. The invention further relates to the use ofsequences from the tomato HMG2 promoter element or its homologs tomodify or construct plant active promoters, which in turn may be used tocontrol the expression of proteins and RNAs in plants and plant cells.

[0030] The tomato HMG2 promoter element and its homologs have a varietyof uses, including but not limited to expressing or overexpressingheterologous genes in a variety of plant cell expression systems, plantcultures, or stably transformed higher plant species. Expression of theheterologous gene product may be induced by elicitor or chemicalinduction in plant cell expression systems, or in response to wounding,pathogen infection, pest infestation as well as elicitor or chemicalinduction in plants.

[0031] The use of the promoter elements described herein to engineerplants may have particular value. By way of illustration, and but bylimitation, an agronomically important plant may be stably transformedwith an HMG2 or HMG2 homolog promoter element or a promoter derivedtherefrom controlling a gene which confers resistance to pathogeninfection or pest infestation. When such a plant is invaded by apathogen or pest, the invasion will trigger the expression of the HMG2or HMG2-homolog promoter element-controlled resistance gene and causethe plant to become increasingly resistant to the pathogen or pest atthe site of invasion.

[0032] The second aspect of the present invention relates to a method ofproducing gene products in harvested plant tissues and plant cellcultures. The method utilizes tissues of transgenic plants ortransformed plant cells engineered with expression constructs comprisinginducible promoters operably linked to the coding sequence of genes ofinterest. Tissues or cultures harvested from engineered plants orculture lines, respectively, may be induced to produce the encodedproteins or RNAs by treatments with the appropriate inducers or inducingconditions.

[0033] As demonstrated by the examples described herein, plants may beengineered with expression constructs comprising an inducible promoter,such as the HMG2 promoter, operably linked to a sequence encoding aprotein or RNA. Tissues harvested from the engineered plants may beinduced to express the encoded protein or RNA by treatment with theappropriate inducer or inducing condition. With the post-harvestproduction method, the amount of encoded gene product produced from anexpression construct utilizing an inducible promoter may surpass thatproduced by an expression construct utilizing a strong constitutivepromoter. Moreover, such harvested plant tissues can maintain, for up totwo weeks after harvesting, their capability for strong inducedproduction of the gene product encoded in the expression construct.

[0034] The post-harvest production method of the invention may beadvantageously used to produce desired gene products that are adverse toplant or plant cell growth or development. In plants or plant cellsengineered with expression constructs comprising inducible promotersoperably linked to sequences encoding the desired gene product, theexpression of the desired but plant-deleterious gene product wouldremain dormant during the growth of the plant or culture line, thusallowing for productive and efficient cultivation or culturing of theengineered plant or culture line. When the engineered plant or cultureline has attained optimal condition for harvest or processing, theexpression of the desired gene product may be artificially triggered bytreatment with the appropriate inducer or inducing condition, which, inthe case with expression constructs comprising the HMG2 promoter, ismechanical wounding or elicitor or chemical induction. The desiredproduct, either encoded by the induced gene or a compound produced bythe induced gene product, may then be extracted from the induced plant,harvested plant tissue, or culture line after allowing for a shortexpression period.

[0035] The post-harvest production method of the invention also may beadvantageously used to produce a labile gene product or a gene productthat synthesizes a labile compound. In plants or plant cells engineeredwith expression constructs comprising inducible promoters operablylinked to sequence encoding the desired gene product, the expression ofthe labile gene product or compound is deferred until the optimalcondition is attained for harvesting of the plant or culture orprocessing of the desired product or compound. The synthesis of thedesired product may then be induced in the plant or culture by treatmentwith the appropriate inducer or inducing condition, and the desired geneproduct or synthesized compound expeditiously extracted from the inducedplant or culture after allowing for a short expression period.

[0036] This aspect of the invention is based in part on the surprisingfindings that harvested plant tissues maintain significant capacity forexpressing inducible genes after excision from the plant and that suchcapacity may actually increase with some storage time.

3.1. Definitions

[0037] The terms listed below, as used herein, will have the meaningindicated.

[0038] 35S=cauliflower mosaic virus promoter for the 35S transcript

[0039] CAT=chloramphenicol acetyltransferase

[0040] cDNA=complementary DNA

[0041] cis-regulatory A promoter sequence 5′ element=upstream of theTATA box that confers specific regulatory response to a promotercontaining such an element. A promoter may contain one or morecis-regulatory elements, each responsible for a particular regulatoryresponse.

[0042] DNA=deoxyribonucleic acid

[0043] functional portion=a functional portion of a promoter is anyportion of a promoter that is capable of causing transcription of alinked gene sequence, e.g., a truncated promoter

[0044] gene fusion=a gene construct comprising a promoter operablylinked to a heterologous gene, wherein said promoter controls thetranscription of the heterologous gene

[0045] gene product=the RNA or protein encoded by a gene sequence

[0046] GUS=1,3-β-Glucuronidase

[0047] heterologous gene=In the context of gene constructs, aheterologous gene means that the gene is linked to a promoter that saidgene is not naturally linked to. The heterologous gene may or may not befrom the organism contributing said promoter. The heterologous gene mayencode messenger RNA (mRNA), antisense RNA or ribozymes.

[0048] HMGR=3-hydroxy-3-methylglutaryl CoA Reductase

[0049] HMG2 homolog=A plant promoter sequence which selectivelyhybridizes to a known HMG2 promoter (e.g., the tomato HMG2 promoterelement disclosed herein), or the promoter of an HMGR gene that displaythe same regulatory responses as an HMG2 promoter (ie. promoter activityis induced by wounding, pathogen-infection, pest-infestation, orelicitor treatment).

[0050] homologous promoter=a promoter that selectively hybridize to thesequence of the reference promoter

[0051] mRNA=messenger RNA

[0052] product of gene product=a product produced by a gene product,e.g., a secondary metabolite

[0053] operably linked=A linkage between a promoter and gene sequencesuch that the transcription of said gene sequence is controlled by saidpromoter.

[0054] RNA=ribonucleic acid

[0055] RNase=ribonuclease.

4. DESCRIPTION OF THE FIGURES

[0056]FIG. 1. The mevalonate/isoprenoid pathway in plants. PGR, plantgrowth regulators.

[0057]FIG. 2. Nucleic acid sequence comparison of tomato HMG2 (SEQ IDNO:1) and tomato HMG1 (SEQ ID NO:2). Sequences are aligned by thealgorithm of Smith and Waterman (Devereux et al., 1984, Nucl. Acid Res.12:387-395) which inserts gaps indicated by dots to optimize alignment.The transcriptional initiation sites are indicated by +1 and arrow. Thetranslational start codons and TATAA boxes are underlined. Thecomparison ends at the first intron of each gene. Ambiguous bases areindicated by lower case letters.

[0058]FIG. 3. Schematic of tomato HMG2 promoter. Panel A The tomato HMG2genomic DNA insert of pTH295. Panel B The 2.5 kb EcoRI fragment ofpTH295 used to general pDW101. pTH295 contains an approximately 7 kbHindIII fragment from the original lambda genomic clone inserted intothe HindIII site of the multiple cloning region of bacterial plasmidpSP6/T7 (BRL). pDW101 contains a 2.5 kb EcoRI fragment of pTH295inserted at the EcoRl site of plasmid bluescript SK-(Stratagene).Restriction endonucleases: Ac, AccI; Av, AvaI; Bg, BglII; E, EcoRI; Ev,EcoRV; H, HindIII; K, Kpnl, P, PstI; T, Taql; X, XbaI. The solid boxesrepresent the four exons encoding HMG2 protein. The fragments belowpDW101 indicate the location of the HMG2 promoter sequences described inFIGS. 4, 5, and 6. The parentheses around HMG2 Sequence 2 indicate thatthe exact location within this region has not yet been determined.

[0059]FIG. 4. Nucleic acid sequence of HMG2 Promoter Sequence I (SEQ IDNO:3). This 1388 bp sequence (SEQ ID NO:3) is from the 3′-end of the 2.5kb EcoRI insert of pDW101. The transcriptional (+1) and translational(ATG) start sites are indicated. The TATAA box, PCR primer locations,and relevant restriction enzyme sites are underlined. Lower case lettersindicate ambiguous bases.

[0060]FIG. 5. Nucleic acid sequence of HMG2 Promoter Sequence II (SEQ IDNO:4). The 480 bp sequence (SEQ ID NO:4) is from an internal region ofthe 2.5 kb EcoRI insert of pDW101. This region spans approximately−1,039 to −2,000 base pairs upstream of the HMG2 translation start site.The exact location of the sequence within this region remains to bedetermined. Two AT repeat motifs are underlined. An X indicates unknownbases; lower case letters are ambiguous bases.

[0061]FIG. 6. Nucleic acid sequence of HMG2 Promoter Sequence III (SEQID NO:5). The 415 bp sequence (SEQ ID NO:5) is from the 5′-end of the2.5 kb EcoRI insert of pDW101. A 23 base palindromic sequence isunderlined. Lower case letters are ambiguous bases.

[0062]FIG. 7. Schematic representations of the promoter deletion clones,pDW201, pDW202 and pDW203, in pBI101. The open bars indicate the insertfragments from the tomato HMG2 promoter. RB and LB are T-DNA bordersequences. The 5′ HindIII and 3′ BamHI sites flanking each HMG2 promoterregion were generated within the sequence of the primers used toPCR-amplify these inserts.

[0063]FIG. 8. Restriction map of plasmid pSLJ330.1. HMGA,EcoRI/BglIIHMG2 promoter fragment from pDW101; GUS, beta glucuronidase codingsequence; ocs 3′, 3′ post-harvest region and polyadenylation sitederived from A. tumefaciens octopine synthetase gene; p35S, promoter ofthe cauliflower mosaic virus 35S transcript; NPT, NPTII gene encodingneomycin phosphotransferase conferring kanamycin resistance; LB, RB,left and right border sequences of Ti plasmids required for transfer ofDNA from Agrobacterium to plant cell.

[0064]FIG. 9. Restriction map of plasmid pSLJ1911. This plasmid was usedto transform plants with a 35S:GUS construct whose activity served as abenchmark to the various HMG2:GUS constructs. Abbreviations are asdescribed in FIG. 8.

[0065]FIG. 10. Tissue specificity of HMG2 Promoter expression intransgenic tobacco and tomato plants. Blue color indicates regions whereGUS is active.

[0066] Panel A. Anthers and pollen of HMG2:GUS compared to analogoustissues from 35S:GUS constructs (plants transformed with pSLJ330.1 orpSLJ1911, respectively).

[0067] Panel B. Trichomes of fully-expanded tobacco leaf (transformationvector pSLJ330.1).

[0068] Panel C. Root section of tomato seedling transformed withpSLJ330.1. Seedling was grown axenically on agar medium. Arrow indicatesthe location of lateral root initiation.

[0069]FIG. 11. Defense-related HMG2 Promoter expression in tobaccotransformed with pSJL330.1.

[0070] Panel A. Leaf tissue 24 (2 left wells) and 48 hours afterwounding and inoculating with water (W, mock) or soft-rotting bacteriumErwinia carotovota ssp. carotovora strain EC14 (EC).

[0071] Panel B. Tobacco seedlings (14 days post germination in sterilepotting mix) 24 and 48 hours following inoculation at site indicated byarrow by placing Rhizoctonia infected oat seed in direct contact withstem.

[0072] Panel C. Leaf section of field grown tobacco showing GUS activitysurrounding necrotic regions due to natural insect predation. Bluepigmentation at the outside edge of disk is due to wound response.

[0073]FIG. 12. Nematode response of HMG2:GUS transgenic tomato(transformed with pSLJ330.1). Tomato seedlings were inoculated withMeloidogyne hapla second stage juveniles. At the indicated times afterinoculation, roots were harvested, incubated overnight in X-Gluc tostain for GUS activity, and subsequently counter-stained with acidfucsin to visualize nematodes.

[0074] Panel A. Squash of root tip 2 days after inoculation.

[0075] Panel B. Root region 3 days post-inoculation. Arrow indicatesregion showing initial GUS activity.

[0076] Panels C, D, & E. Root tips showing characteristicnematode-induced induced swelling 7 days post-inoculation. Note in PanelE that uninfected root tips show no GUS activity.

[0077]FIG. 13. Comparison of post-harvest expression of HMG2promoter-(hatched bars) and CaMV 35S promoter-(open bars) driven GUSgene in transgenic tobacco leaf tissue (transformed with pSLJ330.1 orpSLJ1911, respectively). Leaves were removed from plants at day 0,wound-induced by scoring and stored under moist conditions until harvestat the times indicated. GUS activity was determined as described in thetext.

[0078]FIG. 14: Post-harvest expression of an HMG2:GUS expressionconstruct. The graph shows a comparison of the amount of GUS producedfrom freshly harvested leaves and that from harvested leaves after twoweeks of 4° C. storage. Leaves of mature field-grown plants containingthe HMG2:GUS gene construct were harvested by excising the leaves at thepetiole. The comparison was between the adjacent pairs of leaves fromthe same plant in order to ensure developmental similarity. One set ofleaves, the “FRESH” samples, was processed shortly after harvest bywounding (i.e., by heavily scoring the leaf with a razor blade). Theother set of leaves, the “WEEK 2” samples, was stored in “zip-lock” bagsat 4° C. for two weeks before wounding as described for the freshlyharvested leaves. The GUS activity in the wounded leaves was determinedimmediately after wounding (the “Uninduced” sample) or after 48 hr ofincubation at room temperature (the “48 hr. Induced” sample). GUSactivity is expressed as nM methyl umbelliferone (MUG) released per min.per μg protein. The induced GUS activity from the stored leaf tissue wasconsistently equal to or greater than that from freshly harvested leaftissue. The GUS activity of equivalent fresh leaf tissue of theuntransformed parent cultivar is also shown (the “UNT” sample).

[0079]FIG. 15: The effect of storage on the post-harvest expression ofan HMG2:GUS expression construct. The graph shows a comparison of theamount of GUS produced from harvested leaves that have been stored for 2or 6 weeks at 4° C. or room temperature. Leaves of mature field-growntransgenic tobacco plants containing the HMG2:GUS gene construct wereharvested by excising the leaves at the petiole. Adjacent leaves fromeach plant were sorted into treatment groups, i.e., storage in plasticbags at 4° C. (the “4° C.” samples) or storage at room temperature indry burlap bags (the “Room Temp. Dry” samples). At the times indicated,a sample of the stored leaf tissue was processed by wounding and the GUSactivity in the sample was determined at 0 and 48 hr. after wounding.GUS activity is expressed as nM methyl umbelliferone (MUG) released permin. per μg protein. The induced GUS activity from the stored leaves,even after 6 weeks of storage, was equal to, or higher than, that offreshly harvested leaves.

5. DESCRIPTION OF THE INVENTION

[0080] One aspect of the present invention relates to the HMG2 promoterand its homologs that are involved in the plant response to pathogeninfections, pest infestations or mechanical wounding, and their use todrive the expression of heterologous genes.

[0081] The tomato HMG2 promoter and its homologs control the expressionof a 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) isozyme in plants.HMGR catalyzes the production of mevalonic acid, which is anintermediate in the biosyntheses of various secondary metabolites thathave critical roles in plant defense responses and plant development. Inmost plant tissues, tomato HMG2 promoter and its homologs remain dormantuntil their activity is triggered by pathogen infections, pestinfestations or mechanical wounding. The induction of the HMG2 andhomologous promoters are mediated in part by so called “elicitor”compounds derived from constituents of plant (Darvill and Albersheim,1984, Annu. Rev. Plant Physiol. 35:234) or plant pathogen cell wall orsurface components. When induced, the HMG2 promoter and its homologseffect rapid and strong expression of the genes under their control. Thelack of significant constitutive expression and the rapidly inducible,strong expression characteristics of the HMG2 and homologous promotersmake them ideal elements for controlling the expression of various typesof heterologous genes. Such genes include those encoding pathogen andpest resistance functions, products that are directly or indirectlydeleterious to plant growth, labile products, or products involved inthe biosyntheses of labile compounds, to name but a few.

[0082] According to this aspect of the invention, heterologous genes canbe placed under the control of the tomato HMG2 or homologous promoterelements or promoters derived therefrom, all described herein, and usedto engineer cell culture expression systems, or transgenic plants.Expression of the heterologous gene will be induced in response topathogen infection, pest infestation, mechanical wounding or chemical orelicitor treatment. For example, the induction of the heterologous genein plant cell culture or plant may be induced by treatments with cellwall extracts of plant pathogens, purified elicitors contained withinsuch extracts, or synthetic functional analogs of those elicitors.Alternatively, the expression of the heterologous gene in the plant maybe induced by various bacterial or fungal plant pathogen infections.Similarly, the induction may also be triggered by infestation of theplant by pests such as insects and nematodes.

[0083] The second aspect of the invention relates to a method ofproducing gene products in plant tissues and cell cultures. The methodutilizes the tissues of plants or cultures of plant cells that have beenengineered with expression constructs comprising inducible promotersoperably linked to sequences encoding the desired gene products.According to this aspect of the invention, the production of the desiredgene product takes place in the plant tissue and culture after they havebeen harvested and induced by treatment with the appropriate inducer orinducing condition.

[0084] The invention relates to the family of HMG2 and MG2-derivedpromoters and to the post-harvest production of gene products in planttissues and cultures, and for the purpose of description only, thedescription of the invention will be divided into several stages: (a)inducible promoters that may be used in engineering the plant and plantcells for post-harvest production; (b) isolation, identification andcharacterization of such promoter sequences, including that of the HMG2;(c) identification and characterization of cis-regulatory elementswithin the inducible promoters, including those of the HMG2 promoter,that can regulate other plant promoter sequences; (d) construction ofexpression vectors comprising heterologous genes of interest operablyassociated with an inducible promoters or derivatives thereof, includingexpression vectors comprising HMG2 and derivative promoters; (e)engineering of expression vectors into plants or plant cells; (f)inducing the expression of said expression construct and the productionof the gene product of interest in plant tissues or cell culturesystems; and (g) the types of heterologous gene products that can beadvantageously produced using the post-harvest production method, ormore particularly under the control of the HMG2 promoter elements andHMG2-derived promoters.

[0085] The various embodiments of the claimed invention presented hereinare by the way of illustration and are not meant to limit the invention.

5.1. Inducible Promoters

[0086] The inducible promoters that may be used in the expressionvectors of the post-harvest production method of the invention can beany promoter whose activity is inducible in the host plant. Usefulpromoters include, but are limited to, those whose activities areinduced by chemicals, biological elicitors, heat, cold, salt stress,light, wounding, desiccation, hormone, pathogen infection, orpest-infestation. Table 3 lists some examples of inducible plant geneswhose promoters may be used in the making of the instant invention.

[0087] Useful promoters are any natural or recombinant promoter whoseexpression can be induced in harvested plant tissue. Promoters with sucha capability may be determined using any methods known in the art. Forexample, the promoter may be operably associated with an assayablemarker gene such as GUS; the host plant can be engineered with theconstruct; and the ability and activity of the promoter to drive theexpression of the marker gene in the harvested tissue under variousconditions assayed. TABLE 3 Inducible Plant Genes with Potential forPost-harvest Induction and Accumulation of Transgene Products. Anasterisk designates those genes for which promoters have been isolatedand characterized. References represent one to two representativereferences or relevant review article and are not intended to beexhaustive. Genes/gene products Functions Sources of clonesDefense-Response Genes^(a1) Phytoalexin biosynthesis Phenylpropanoidphytoalexin *Phenylalanine ammonia lyase (PAL)² Enzyme, central pathwayBean, parsley, potato, tomato 4-Coumarate CoA ligase (4CL)³ Enzyme,central pathway Parsley, potato *Chalcone synthase (CHS)^(4,5) Enzyme,Isoflavanoid branch Bean, soybean, parsley Chalcone isomerase (CHI)⁶Enzyme, Isoflavanoid branch Bean Resveratrol (stilbene) synthase⁷Enzyme, Isoflavanoid branch Grapevine, peanut Isoflavone reductase(IFR)⁸ Enzyme, Isoflavanoid branch Alfalfa Terpenoid phytoalexins*HMG-CoA reductase (HMG)^(9,10) Enzymes, central pathway Tomato,tobacco, potato, rice Casbene synthetase¹¹ Casbene biosynthesis Castorbean Cell wall components Lignin *Phenylalanine ammonia lyase See aboveCinnamyl alcohol dehydrogenase (CAD)¹² Lignin biosyn. Tobacco Caffeicacid o-methyltransferase¹³ Lignin biosyn. Alfalfa, tobaccoLignin-forming peroxidase¹⁴ Lignin polymerization Tobacco, wheatHydroxyproline-rich glycoproteins (HRGP)^(15,16) Structural proteinBean, tomato Glycine-rich proteins (GRP)¹⁵ Structural protein Bean,potato, pea, rice Thionins¹⁷ Antifungal Barley Hydrolases, lytic enzymes*Chitinases (PR-P, PR-Q)¹⁸⁻²⁰ Class I chitinase, basic Vacuolar,antifungal Tobacco, bean, tomato Class I and II chitinase, acidicExtracellular, antifungal Bean Class II chitinase Bifunctional lysozyme,Cucumber, tobacco, chitinase barley, petunia *β-1,3-Glucanase²¹Antifungal, chitinase Bean, tobacco, synergist potato, pea, rice,Arabidopsis β-fructosidase²² Antifungal invertase Tomato Others*Proteinase inhibitors (PI-I, PI-II)^(23,24) Trypsin-, chymotrypsin-Potato, tomato inhibitors Superoxide dismutase (SOD)²⁵ Anti-oxidantenzyme Tobacco, maize, tomato Lipoxygenase²⁶ Lipid peroxidation,Arabidopsis jasmonate biosyn. Additional “pathogenesis-related” prot.*PRI family, PR2, PR3²⁷⁻²⁹ Unknown Tobacco, bean, parsley, pea Osmotin,PR5³⁰⁻³² Antifungal, thaumatin-like Tobacco, maize Ubiquitin³³ Proteindegradation Potato Wound-Inducible Genes^(a) *win1, *win2(hevein-like)³⁴ Chitin-binding prot. Potato (hevein, rubber tree) wun1,wun2³⁵ Unknown Potato *nos, nopaline synthase³⁶ Agrobacterium nutr.Agrobacterium tumefaciens ACC synthase³⁷ Ethylene biosynthesis Tomato,squash HMG-CoA reductase hmg1³⁸ Sterol/alkaloid synth. Potato3-deoxy--D-arabino-heptulosonate- Lignin biosyn. Potato, tomato7-phosphate synthase³⁹ HSP70³³ Heat-shock protein, Potato chaparoneSalicylic acid inducible⁴⁰ acid peroxidase¹⁴ Lignin-forming TobaccoPR-proteins^(40,41) (see above) Tobacco Glycine-rich protein⁴¹ Cell wallprotein Tobacco Methyl jasmonate inducible *vspB⁴² Vacuolar storageprot. Soybean Proteinase inhibitors I and II⁴³ Trypsin, chymotrypsininhib. Potato, tomato Heat-shock genes⁴³ HSP70³³ Chaperonin PotatoUbiquitin (see above) Cold-stress inducible⁴⁴ Drought, salt stress⁴⁵Osmotin³⁰⁻³² Desiccation tolerance Tobacco, maize Hormone inducibleGibberellin α-amylase⁴⁶ Starch degradation Barley Abscisic acid^(45,47)EM-1, RAB, LEA genes⁴⁵ Unknown, embryogenesis Wheat, rice, maize, cottonEthylene Chitinase, phytoalexin biosyn. genes (see above)

[0088] Useful promoters may be tissue-specific. Non-tissue-specificpromoters (i.e., those that express in all tissues after induction),however, are preferred. More preferred are promoters that additionallyhave no or very low activity in the uninduced state. Most preferred arepromoters that additionally have very high activity after induction.

[0089] In a particular embodiment, the inducible promoter is the HMG2promoter and derivatives thereof. The HMG2 and derivative promoters,which in addition to having utility in the production method of theinvention, also have utility in the HMG2 promoter expression system ofthe invention. This aspect of the invention is also described herein insome detail.

5.2. Promoter Isolation and Characterization

[0090] According to the present invention, functional portions of thepromoters described herein refer to regions of the nucleic acid sequencewhich are capable of promoting transcription of an operably linked genein response to an inducer or inducing condition at some point in thelife cycle of the plant or cell culture, including after the tissue orculture has been harvested. In the case of the HMG2 promoter, thefunctional portions are those regions which are capable of causingtranscription of an operably linked gene in response to wounding,pathogen infection, pest infestation or chemical/elicitor treatmentduring the entire pattern of plant development.

[0091] Homologous nucleotide sequences is used herein to mean nucleicacid sequences which are capable of selectively hybridizing to eachother. Selectively hybridizing is used herein to mean hybridization ofDNA or RNA probes 50 bases or greater in length from one sequence to the“homologous” sequence under stringent conditions, e.g., washing in0.1×SSC/0.1% SDS at 68° C. for at least 1 hour (Ausubel, et al., Eds.,1989, Current Protocols in Molecular Biology, Vol. I, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York, at page 2.10.3)

[0092] Nucleotide sequences homologous to the tomato HMG2 promoterdescribed herein refers to nucleic acid sequences which are capable ofselectively hybridizing to the nucleic acid sequence contained in theapproximately 2.2 kb EcoRI-Bql II fragment of pDW101 (NRRL Accession No.______) in hybridization assays or which are homologous by sequenceanalysis (containing a span of 100 basepairs in which at least percentof the nucleotides are identical to the sequences presented herein) andwhich has promoter activity identical or very similar to that of HMG2,i.e., wound, elicitor, pathogen, etc., inducibility. Such a promoter maybe precisely mapped and its activity may be ascertained by methods knownin the art, e.g., deletion analysis, expression of marker genes, RNaseprotection, and primer extension analysis of mRNAs preparations fromplant tissues containing the homologous promoter operably linker to amarker gene.

[0093] Homologous nucleotide sequences refer to nucleotide sequencesincluding, but not limited to, natural promoter elements in diverseplant species as well as genetically engineered derivatives of thepromoter elements described herein.

[0094] Methods which could be used for the synthesis, isolation,molecular cloning, characterization and manipulation of the induciblepromoter sequences, including that of the HMG2 promoter, describedherein are well known to those skilled in the art. See, e.g., thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989).

[0095] According to the present invention, the inducible promotersequences, including that of the HMG2 promoter, or portions thereofdescribed herein may be obtained from an appropriate plant source, fromcell lines or recombinant DNA constructs containing the induciblepromoter sequences or genes sequences comprising the promoter sequences,and/or by chemical synthetic methods where the promoter sequence isknown. For example, chemical synthetic methods which are well known tothose skilled in the art can be used to synthesize the HMG2 sequencesdepicted in FIGS. 4, 5 and 6 herein (SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, respectively). The synthesized sequences can be cloned andexpanded for use as promoter elements. Such synthetic sequences can alsobe used as hybridization probes to identify and isolate the desiredpromoter sequences from appropriate plant and cellular sources.

[0096] Alternatively, a desired promoter element may be identified andisolated by screening gene libraries. For example, a genomic DNA librarymay be screened for clones containing sequences homologous to knowninducible genes, e.g., HMGR genes or, alternatively, inducible promotersequences, e.g., HMGR promoters. For example, sequences from genomic orcDNA clones encoding the inducible gene or oligonucleotide probescorresponding to known amino acid sequences of the inducible protein maybe used as hybridization probes to identify homologous clones in agenomic DNA library using standard methods. Such methods include, forexample, the method set forth in Benton and Davis (Science 196:180(1977)) for bacteriophage libraries, and Grunstein and Hogness (Proc.Nat. Acad. Sci. USA 72:3961-3965 (1975)) for plasmid libraries.

[0097] In instances where the species sources of the probe and thelibrary are widely divergent, such as between a monocot and a dicot, theprobe is preferably from a region encoding highly conserved amino acidresidues of the inducible protein and the hybridization and wash arecarried out at moderate stringencies, e.g., washing in 0.2×SSC/0.1% SDSat 42° C. (Ausubel, et al., 1989, supra). Homologous gene sequences arethose that are detected by the probe under such conditions and thatencode a protein with 75% or greater amino acid homology with theprotein encoded by the gene that is the source of the probe. Inisolating HMG2 homologous genes, useful hybridization conditions havebeen described in detail. See Park et al., 1992, Plant Mol. Biol.20:327-331.

[0098] Clones containing homologous inducible gene sequences may bedistinguished from other isogenes by their ability to detect, at highstringency hybridization conditions, mRNA transcripts that are inducedby the expected inducing factor or condition. For example, HMG2homologous genes may be identified by the rapid appearance ofhybridizing mRNA transcripts following wounding, pathogen infection,pest-infestation, or elicitor treatment (for an example of such ananalysis see Yang et al., 1991, Plant Cell 3:397-405). The HMG2 clonesmay also be identified by examining their ability to hybridize atmoderate or high stringency condition to 5′ untranslated region or 5′upstream region of known HMG1 and HMG2 (e.g., the 2.5 kb EcoRI fragmentdepicted in FIG. 3) genes. HMG2 clones can be identified as those thatshow a stronger signal with the HMG2 probe than with the HMG1 probe.

[0099] In other instances, where the species sources of the probe andthe library are not widely divergent, such as between closely relatedplants, the probes used to screen such libraries may consist ofrestriction fragments or nucleotide sequences encoding the N-terminal200 or so amino acids of the inducible protein, e.g., in the case oftomato HMG2 HMGR see Park et al., 1992, Plant Mol. Biol. 20:327-331,portions thereof or nucleotide sequences homologous thereto. Retrievedclones may then be analyzed by restriction fragment mapping andsequencing techniques according to methods well known in the art.

[0100] In another approach, restriction fragments or nucleotidesequences of the inducible promoter or portions thereof and sequenceshomologous thereto may be used to screen genomic libraries to identifygenomic clones containing homologous promoter sequences using thestandard techniques described supra.

[0101] In an embodiment, the tomato HMG2 promoter elements described inFIG. 3, FIG. 4, FIG. 5, and FIG. 6, portions thereof and sequenceshomologous thereto may be used to screen genomic libraries to identifygenomic clones containing homologous promoter sequences. For example,the 2.5 kb EcoRI fragment containing the tomato HMG2 promoter in plasmidpDW101 or portions thereof may be used to such ends.

[0102] In the above two approaches, the hybridization and washconditions used to identify clones containing the homologous promotersequences may be varied depending on the evolutionary relatednessbetween the species origins of the probe and the genomic libraryscreened. The more distantly related organisms would require moderatehybridization and wash conditions. See supra for two wash conditionsthat can be used.

[0103] In yet another approach to the isolation of inducible promoterelements, known oligonucleotide sequences of inducible promoters can beused as primers in PCR (polymerase chain reactions) to generate thepromoter sequences from any plant species. Similar to the hybridizationapproaches, the amplification protocol may be adjusted according to theevolutionary relatedness between the target organism and primer-sourceorganism. The adjustments may include the use of degenerate primers andprotocols well known in the art for amplifying homologous sequences. Fora review of PCR techniques, see for example, Gelfind, 1989, PCRTechnology. Principles and Applications for DNA Amplification, Ed., H.A. Erlich, Stockton Press, N.Y., and Current Protocol in MolecularBiology, Vol. 2, Ch. 15, Eds. Ausubel et al., John Wiley & Sons, 1988.

[0104] In instances where only internal sequences of an induciblepromoter are known, sequences flanking such sequences may be obtained byinverse PCR amplification (Triglia et al., 1988, Nucl. Acids Res.16:8186). The flanking sequences can then be linked to the internalpromoter sequences using standard recombinant DNA methods in order toreconstruct a sequence encompassing the entirety of the induciblepromoter.

[0105] The location of the inducible promoter within the sequencesisolated as described above may be identified using any method known inthe art. For example, the 3′-end of the promoter may be identified bylocating the transcription initiation site, which may be determined bymethods such as RNase protection (Liang et al., 1989, J. Biol. Chem.264:14486-14498), primer extension (Weissenborn and Larson, 1992, J.Biol. Chem. 267:6122-6131), reverse transcriptase/PCR. The location ofthe 3′-end of the promoter may be confirmed by sequencing and computeranalysis, examining for the canonical AGGA or CAT and TATA boxes ofpromoters that are typically 50-60 base pairs (bp) and 25-35 bp5′-upstream of the transcription initiation site. The 5′-end promotermay be defined by deleting sequences from the 5′-end of the promotercontaining fragment, constructing a transcriptional or translationalfusion of the resected fragment and a reporter gene, and examining theexpression characteristics of the chimeric gene in transgenic plants.Reporter genes that may be used to such ends include, but are notlimited to, GUS, CAT, luciferase, β-galactosidase and C1 and R genecontrolling anthocyanin production.

[0106] According to the present invention, an inducible promoter elementis one that confers to an operably linked gene in a plant or plantcell: 1) minimal expression in most plant tissues; and 2) inducedexpression following treatment by inducer or inducing condition known totrigger the expression of the promoter or gene from which the element isderived from.

[0107] In an embodiment, the HMG2 promoter element is one that confersto an operably linked gene in a plant or plant cell: 1) minimalexpression in most plant tissues; and 2) induced expression followingwounding, pathogen infection, pest infestation or chemical/elicitortreatment. An HMG2 promoter element may additionally confer specificdevelopmental expression as in pollen, mature fruit tissues, and roottissues of the zone of lateral root initiation. See Table 1, whichcompares the expression characteristics of tomato HMG2 promoter to theother known plant HMGR promoters.

[0108] According to the present invention, a promoter element comprisethe region between −2,500 bp and +1 bp upstream of the transcriptioninitiation site of inducible gene, or portions of said region. In aparticular embodiment, the HMG2 promoter element comprises the regionbetween positions −2,300 and +1 in the 5′ upstream region of the tomatoHMG2 gene (see FIG. 3). Another embodiment of the HMG2 promoter elementcomprises the region between positions −891 and +1 in the 5′ upstreamregion of the tomato HMG2 gene. An additional embodiment of HMG2promoter element comprises the region between positions −347 and +1 inthe 5′ upstream region of the tomato HMG2 gene. Yet another embodimentof HMG2 promoter element comprises the region between positions −58 and+1 in the 5′ upstream region of the tomato HMG2 gene. In furtherembodiments of the present invention, an HMG2 promoter element maycomprise sequences that commence at position +1 and continues 5′upstream up to and including the whole of the nucleotide sequencedepicted in FIG. 4, FIG. 5 or FIG. 6.

5.3. Cis-Regulatory Elements of Promoters

[0109] According to the present invention, the cis-regulatory elementswithin an inducible promoter may be identified using any method known inthe art. For example, the location of cis-regulatory elements within aninducible promoter may be identified using methods such as DNase orchemical footprinting (Meier et al., 1991, Plant Cell 3:309-315) or gelretardation (Weissenborn and Larson, 1992, J. Biol. Chem. 267-6122-6131;Beato, 1989, Cell 56:335-344; Johnson et al., 1989, Ann. Rev. Biochem.58:799-839). Additionally, resectioning experiments may also be employedto define the location of the cis-regulatory elements. For example, aninducible promoter-containing fragment may be resected from either the5′ or 3′-end using restriction enzyme or exonuclease digests.

[0110] To determine the location of cis-regulatory elements within thesequence containing the inducible promoter, the 5′- or 3′-resectedfragments, internal fragments to the inducible promoter containingsequence, or inducible promoter fragments containing sequencesidentified by footprinting or gel retardation experiments may be fusedto the 5′-end of a truncated plant promoter, and the activity of thechimeric promoter in transgenic plant examined as described in section5.2. above. Useful truncated promoters to these ends comprise sequencesstarting at or about the transcription initiation site and extending tono more than 150 bp 5′ upstream. These truncated promoters generally areinactive or are only minimally active. Examples of such truncated plantpromoters may include, among others, a “minimal” CaMV 35S promoter whose5′ end terminates at position −46 bp with respect to the transcriptioninitiation site (Skriver et al., Proc. Nat. Acad. Sci. USA88:7266-7270); the truncated “−90 35S” promoter in the X-GUS-90 vector(Benfey and Chua, 1989, Science 244:174-181); a truncated “−101 nos”promoter derived from the nopaline synthase promoter (Aryan et al.,1991, Mol. Gen. Genet. 225:65-71); and the truncated maize Adh-1promoter in pADcat 2 (Ellis et al., 1987, EMBO J. 6:11-16).

[0111] According to the present invention, a cis-regulatory element ofan inducible promoter is a promoter sequence that can confer to atruncated promoter one or more of the inducible properties of theoriginal inducible promoter. For example, an HMG2 cis-regulatory elementis an HMG2 promoter sequence that can confer to a truncated promoter oneor more of the following characteristics in expressing an operablylinked gene in a plant or plant cell: 1) induced expression followingwounding; 2) induced expression following pathogen infection; 3) inducedexpression following pest infestation; 4) induced expression followingchemical elicitor treatment; or 5) expression in pollen, mature fruit,or other developmentally defined tissues identified as expressing HMG2.Further, an HMG2 cis-regulatory element may confer, in addition to theabove described characteristics, the ability to suppress theconstitutive activity of a plant promoter.

5.4. Inducible Promoter-Driven Expression Vectors

[0112] The properties of the nucleic acid sequences are varied as arethe genetic structures of various potential host plant cells. Thepreferred embodiments of the present invention will describe a number offeatures which an artisan may recognize as not being absolutelyessential, but clearly advantageous. These include methods of isolation,synthesis or construction of gene constructs, the manipulation of thegene constructs to be introduced into plant cells, certain features ofthe gene constructs, and certain features of the vectors associated withthe gene constructs.

[0113] Further, the gene constructs of the present invention may beencoded on DNA or RNA molecules. According to the present invention, itis preferred that the desired, stable genotypic change of the targetplant be effected through genomic integration of exogenously introducednucleic acid construct(s), particularly recombinant DNA constructs.Nonetheless, according to the present inventions, such genotypic changescan also be effected by the introduction of episomes (DNA or RNA) thatcan replicate autonomously and that are somatically and germinallystable. Where the introduced nucleic acid constructs comprise RNA, planttransformation or gene expression from such constructs may proceedthrough a DNA intermediate produced by reverse transcription.

[0114] The present invention provides for use of recombinant DNAconstructs which contain inducible promoter fragments, functionalportions thereof, and nucleotide sequences homologous thereto. As usedherein a functional portion of a promoter and a promoter homologoussequence are both capable of functioning as an inducible promoter. Thefunctionality of such sequences can be readily established by any methodknown in the art. Such methods include, for example, constructingexpression vectors with such sequences and determining whether theyconfer inducible expression to an operably linked gene. In particularembodiments, the invention provides for the use of tomato HMG2 promoterfragments or sequences as depicted in FIGS. 3, 4, 5, and 6, functionalportions thereof, and nucleotide sequences homologous thereto.

[0115] The inducible promoter elements of the invention may be used todirect the expression of any desired protein, or to direct theexpression of an RNA product, including, but not limited to, an“antisense” RNA or ribozyme. Such recombinant constructs generallycomprise a native inducible promoter or a recombinant inducible promoterderived therefrom, ligated to the nucleic acid sequence encoding adesired heterologous gene product.

[0116] A recombinant inducible promoter is used herein to refer to apromoter that comprises a functional portion of a native induciblepromoter or a promoter that contains native promoter sequences that ismodified by a regulatory element from a inducible promoter. For example,in particular embodiments, a recombinant inducible promoter derived fromthe HMG2 promoter may comprise the approximately 0.17 kb, 0.46 kb or 1.0kb HindIII-BamHI tomato HMG2 promoter fragment of pDW201, pDW202 andpDW203, respectively (see FIG. 7). Alternatively, a recombinantinducible promoter derived from the HMG2 promoter may be a chimericpromoter, comprising a full-length or truncated plant promoter modifiedby the attachment of one or more HMG2 cis-regulatory elements.

[0117] The manner of chimeric promoter constructions may be any wellknown in the art. For examples of approaches that can be in suchconstructions, see section 5.3. above and Fluhr et al., 1986, Science232:1106-1112; Ellis et al., 1987, EMBO J. 6:11-16; Strittmatter andChua, 1987, Proc. Nat. Acad. Sci. USA 84:8986-8990; Poulsen and Chua,1988, Mol. Gen. Genet. 214:16-23; Comai et al., 1991, Plant Molec. Biol.15:373-381; Aryan et al., 1991, Mol. Gen. Genet. 225:65-71.

[0118] According to the present invention, where an inducible promoteror a recombinant inducible promoter is used to express a desiredprotein, the DNA construct is designed so that the protein codingsequence is ligated in phase with the translational initiation codondownstream of the promoter. Where the promoter fragment is missing5′-leader sequences, a DNA fragment encoding both the protein and its 5′RNA leader sequence is ligated immediately downstream of thetranscription initiation site. Alternatively, an unrelated 5′ RNA leadersequence may be used to bridge the promoter and the protein codingsequence. In such instances, the design should be such that the proteincoding sequence is ligated in phase with the initiation codon present inthe leader sequence, or ligated such that no initiation codon isinterposed between the transcription initiation site and the firstmethionine codon of the protein.

[0119] Further, it may be desirable to include additional DNA sequencesin the protein expression constructs. Examples of additional DNAsequences include, but are not limited to, those encoding: a 3′untranslated region; a transcription termination and polyadenylationsignal; an intron; a signal peptide (which facilitates the secretion ofthe protein); or a transit peptide (which targets the protein to aparticular cellular compartment such as the nucleus, chloroplast,mitochondria, or vacuole).

5.5. Recombinant DNA Constructs

[0120] The recombinant construct of the present invention may include aselectable marker for propagation of the construct. For example, aconstruct to be propagated in bacteria preferably contains an antibioticresistance gene, such as one that confers resistance to kanamycin,tetracycline, streptomycin, or chloramphenicol. Suitable vectors forpropagating the construct include plasmids, cosmids, bacteriophages orviruses, to name but a few.

[0121] In addition, the recombinant constructs may includeplant-expressible, selectable, or screenable marker genes for isolating,identifying or tracking plant cells transformed by these constructs.Selectable markers include, but are not limited to, genes that conferantibiotic resistance, (e.g., resistance to kanamycin or hygromycin) orherbicide resistance (e.g., resistance to sulfonylurea,phosphinothricin, or glyphosate). Screenable markers include, but arenot be limited to, genes encoding β-glucuronidase (Jefferson, 1987,Plant Molec Biol. Rep 5:387-405), luciferase (Ow et al., 1986, Science234:856-859), B protein that regulates anthocyanin pigment production(Goff et al., 1990, EMBO J 9:2517-2522).

[0122] In embodiments of the present invention which utilize theAgrobacterium tumefacien system for transforming plants (see infra), therecombinant constructs may additionally comprise at least the rightT-DNA border sequences flanking the DNA sequences to be transformed intothe plant cell. Alternatively, the recombinant constructs may comprisethe right and left T-DNA border sequences flanking the DNA sequence. Theproper design and construction of such T-DNA based transformationvectors are well known to those skill in the art.

5.6. Production of Transgenic Plants and Plant Cells

[0123] According to the present invention, a desirable plant or plantcell may be obtained by transforming a plant cell with the nucleic acidconstructs described herein. In some instances, it may be desirable toengineer a plant or plant cell with several different gene constructs.Such engineering may be accomplished by transforming a plant or plantcell with all of the desired gene constructs simultaneously.Alternatively, the engineering may be carried out sequentially. That is,transforming with one gene construct, obtaining the desired transformantafter selection and screening, transforming the transformant with asecond gene construct, and so on.

[0124] In an embodiment of the present invention, Agrobacterium isemployed to introduce the gene constructs into plants. Suchtransformations preferably use binary Agrobacterium T-DNA vectors(Bevan, 1984, Nuc. Acid Res. 12:8711-8721), and the co-cultivationprocedure (Horsch et al., 1985, Science 227:1229-1231). Generally, theAgrobacterium transformation system is used to engineer dicotyledonousplants (Bevan et al., 1982, Ann. Rev. Genet 16:357-384; Rogers et al.,1986, Methods Enzymol. 118:627-641). The Agrobacterium transformationsystem may also be used to transform as well as transfer DNA tomonocotyledonous plants and plant cells. (see Hernalsteen et al., 1984,EMBO J 3:3039-3041 ; Hooykass-Van Slogteren et al., 1984, Nature311:763-764; Grimsley et al., 1987, Nature 325:1677-179; Boulton et al.,1989, Plant Mol. Biol. 12:31-40.; Gould et al., 1991, Plant Physiol.95:426-434).

[0125] In other embodiments, various alternative methods for introducingrecombinant nucleic acid constructs into plants and plant cells may alsobe utilized. These other methods are particularly useful where thetarget is a monocotyledonous plant or plant cell. Alternative genetransfer and transformation methods include, but are not limited to,protoplast transformation through calcium-, polyethylene glycol (PEG)-or electroporation-mediated uptake of naked DNA (see Paszkowski et al.,1984, EMBO J 3:2717-2722, Potrykus et al. 1985, Molec. Gen. Genet.199:169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828;Shimamoto, 1989, Nature 338:274-276) and electroporation of planttissues (D'Halluin et al., 1992, Plant Cell 4:1495-1505). Additionalmethods for plant cell transformation include microinjection, siliconcarbide mediated DNA uptake (Kaeppler et al., 1990, Plant Cell Reporter9:415-418), and microprojectile bombardment (see Klein et al., 1988,Proc. Nat. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al., 1990, PlantCell 2:603-618).

[0126] According to the present invention, a wide variety of plants andplant cell systems may be engineered for the desired physiological andagronomic characteristics described herein using the nucleic acidconstructs of the instant invention and the various transformationmethods mentioned above. In preferred embodiments, target plants andplant cells for engineering include, but are not limited to, those ofmaize, wheat, rice, soybean, tomato, tobacco, carrots, peanut, potato,sugar beets, sunflower, yam, Arabidopsis, rape seed, and petunia.

5.7. Selection and Identification of Transformed Plants and Plant Cells

[0127] According to the present invention, desired plants and plantcells may be obtained by engineering the gene constructs describedherein into a variety of plant cell types, including but not limited to,protoplasts, tissue culture cells, tissue and organ explants, pollen,embryos as well as whole plants. In an embodiment of the presentinvention, the engineered plant material is selected or screened fortransformants (i.e., those that have incorporated or integrated theintroduced gene construct(s)) following the approaches and methodsdescribed below. An isolated transformant may then be regenerated into aplant. Alternatively, the engineered plant material may be regeneratedinto a plant or plantlet before subjecting the derived plant or plantletto selection or screening for the marker gene traits. Procedures forregenerating plants from plant cells, tissues or organs, either beforeor after selecting or screening for marker gene(s), are well known tothose skilled in the art.

[0128] A transformed plant cell, callus, tissue or plant may beidentified and isolated by selecting or screening the engineered plantmaterial for traits encoded by the marker genes present on thetransforming DNA. For instance, selection may be performed by growingthe engineered plant material on media containing inhibitory amounts ofthe antibiotic or herbicide to which the transforming marker geneconstruct confers resistance. Further, transformed plants and plantcells may also be identified by screening for the activities of anyvisible marker genes (e.g., the β-glucuronidase, luciferase, B or C1genes) that may be present on the recombinant nucleic acid constructs ofthe present invention. Such selection and screening methodologies arewell known to those skilled in the art.

[0129] Physical and biochemical methods also may be used to identify aplant or plant cell transformant containing the gene constructs of thepresent invention. These methods include but are not limited to: 1)Southern analysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) northern blot, S-1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins; 5) biochemical measurements of compounds produced as aconsequence of the expression of the introduced gene constructs.Additional techniques, such as in situ hybridization, enzyme staining,and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the arts.

5.8. Expression of Heterologous Gene Products in Transgenic Plants

[0130] The present invention may be advantageously used to direct theexpression of a variety of gene products. These gene products include,but are not limited to, proteins, anti-sense RNA and ribozymes.

[0131] In embodiments of the present invention, a inducible promoter ora recombinant inducible promoter may be used, in post-harvestproduction, to express in plants and plant cell cultures a variety ofhigh valued protein products, including, but not limited to,pharmaceutical and therapeutic enzymes and proteins. Such proteinproducts may include, for example, various peptide-hormones, cytokines,growth factors, antibodies, blood proteins and vaccines. Further, thesepromoter elements may also be used to express in plants and cellcultures multiple enzymes of complex biosynthetic pathways, theinduction of which would confer to host plant or plant cell the abilityto produce complex biochemicals and biologicals. Examples of suchproducts include secondary metabolites such as alkaloids, antibiotics,pigments, steroids and complex biological structures such as intact ordefective viruses or viral particles. Additionally, these promoterelements may also be used to express various types of lytic andprocessing enzymes that can convert what would be otherwise unusable orlow quality plant compounds or constituents into useful or high qualitycompounds or chemical feedstocks. Examples of such products includecellulases, lignases, amylases, proteases, pectinases, phytases, etc.Furthermore, a inducible promoter or a recombinant inducible promotermay also be used to express RNA products such as antisense RNA andribozymes. In particular embodiments, HMG2 or HMG2-derived promoterelements which confer wound-inducible and/or elicitor-inducible geneexpression are used to express the above-mentioned products.

[0132] In the above embodiments, the inducible promoter or recombinantinducible promoter, including the wound- and/or elicitor-specific HMG2and HMG2-derived promoters, may be advantageously used to direct the“post-harvest” production and accumulation of the desired direct orindirect gene products. That is, the production of the desired productsdoes not occur during normal growth of the plant or the cell culture,but only occurs after the plant or the cell culture (e.g., callusculture) is mechanically macerated and/or elicitor-treated shortlybefore, during, or shortly after, harvesting the plant or cell cultures.(For a general reference describing plant cell culture techniques thatcould be used in conjunction with the “post-harvest” production approachdisclosed here, see Handbook of Plant Cell Culture, Vol. 4, Techniquesand Applications, etc., Evans, D. A., Sharp, W. R. and Ammirato, P. V.,1986 Macmillan Publ., New York, N.Y.).

[0133] Any plant tissue or culture lines of plants or plant cellsengineered with the expression construct described herein may used inthe production of desired direct or indirect gene products. Useful planttissue (and organs) include, but are not limited to, leaf, stem, root,flower, fruit and seed.

[0134] Where the production of the desired indirect gene product, e.g.,a secondary metabolite, requires two or more gene functions, it may beadvantageous to engineer the host plant or plant cell with severalexpression constructs, wherein each construct comprises the sameinducible promoter controlling the expression of each required genefunction, so as to enable the coordinate expression of all required genefunctions.

[0135] The induction of the harvested tissues and cultures from theengineered plants and cells may be by any means known in the art for theparticular inducible promoter used in the expression construct. Forexample, to induce expression from an expression construct vectorcomprising an HMG2 or HMG2-derived promoter, the harvested tissue orculture may be physically wounded by maceration, treated with biologicalelicitors, infected with an appropriate pathogen, etc.

[0136] The induction of the harvested plant tissue or culture may bedone immediately after harvesting or the harvested tissue or culture maybe stored and then subsequently induced. The storage of the harvestedplant tissue or culture may be by any procedure or method known in theart that optimally preserves the gene expression capability of thestored material.

[0137] In particular embodiments, harvested leaves can be stored at 4°C. in sealed containers or at room temperature in air permeablecontainers for up to 6 weeks before inducing the expression of thedesired gene products. Preferably, the stored leaves are induced atabout two weeks after harvest.

[0138] The induced tissue or culture may be incubated at roomtemperature for up to a week to allow for the expression of the inducedgene(s) and accumulation of the desired direct or indirect gene productbefore the tissue or culture is processed for isolating the desireddirect or indirect gene product. In particular embodiments, induced leaftissue is incubated at room temperature for approximately 48 hr. beforethe leaf tissue is processed.

[0139] The application of inducible promoter or recombinant induciblepromoters to produce the above mentioned types of products isparticularly significant given that many such products may be eitherlabile or deleterious to cellular metabolism or plant growth. Thus,their efficient and optimal production, in many instances, may be bestachieved by the use of inducible, particularly wound-inducible orelicitor-inducible, promoters coupled with a post-harvest inductionprotocol. As explained previously, harvesting of plants or plant partsfor later use does not normally kill or harm plant tissues if they aremaintained in an environmentally suitable condition.

[0140] In another embodiment, an HMG2 or HMG2-derived promoter whichconfers pathogen-induced expression may be used to express a variety ofdisease resistance genes during a pathogen infection. Examples of suchresistance genes include virus coat protein, anti-sense RNA or ribozymegenes for anti-virus protection (Gadani et al., 1990, Arch. Virol115:1-21); lysozymes, cecropins, maganins, or thionins foranti-bacterial protection; or the pathogenesis-related (PR) proteinssuch as glucanases and chitinases for anti-fungal protection.

[0141] In a further embodiment, an HMG2 or HMG2-derived promoter whichconfers pest infestation-induced expression may be used to express avariety of pest resistance genes during an insect or nematodeinfestation. Examples of useful gene products for controlling nematodesor insects include Bacillus thuringiensis endotoxins, proteaseinhibitors, collagenases, chitinase, glucanases, lectins, glycosidases,and neurotoxins.

[0142] The HMG2 and HMG2-derived promoters have a number ofcharacteristics that make them particularly useful for expressingpathogen and pest resistance genes. These characteristics include thesepromoters' very low background activity in most uninduced tissues of theplant, their rapid induction and strong activity once induced and therelatively site-specific nature of the induced response. These combinedcharacteristics make the HMG2-controlled resistance functions highlyefficient by limiting the resistance response to the time and place thatsuch plant responses would be the most effective (i.e., the site of thepathogen or pest ingress).

[0143] In yet another embodiment, an HMG2 or HMG2-derived promoter whichconfers pollen-specific expression may be used to engineer male-sterileplants. A pollen-specific HMG2 or HMG2-derived promoter may be used toexpress gene functions that interfere with vital-cellular processes suchas gene expression, cell division or metabolism. Examples of suchfunctions include RNases, DNases, anti-sense RNAs and ribozymes. The useof pollen-specific HMG2 or HMG2-derived promoters would limit theexpression of such deleterious function to pollen tissue withoutaffecting other aspects of normal plant growth development.

6.0. EXAMPLE Isolation and Characterization of the Tomato HMG2 Promoterand HMG2 Gene Fusions

[0144] The isolation of the tomato HMG2 promoter is described here. Thedisclosed approach is generally applicable to the isolation of homologsof any cloned promoter, and is particularly applicable to the isolationof homologs of the tomato HMG2 promoter, from other plant species. Theapproach uses a gene sequence containing the coding region of an HMGR toscreen a genomic library under low stringency hybridization conditions.In the present example, the probe used was a fragment containing aregion of the yeast HMGR that shows a high degree of amino acid sequenceconservation with the hamster HMGR. Positive clones are isolated andsubcloned where necessary, and the hybridizing region is sequenced alongwith the flanking sequences. Sequences containing the HMG2 gene areidentified based on their nucleotide sequence comparisons with knownHMGR genes and their divergence from known HMG1 genes (see Park et al.,1992, Plant Mol. Biol. 20:327-331).

[0145] Promoter:reporter gene fusions are used here to localize the HMG2promoter element within the cloned sequence 5′ upstream of the HMGRcoding sequence. One goal of the analyses is to demonstrate thetissue-specificity and defense-related and post-harvest inducibilitythat the 2.3 kb HMG2 promoter confers on operably linked heterologousgenes. The second goal of the analysis is to determine in transgenicplants the smallest 5′ upstream fragment that would confer the completearray of regulatory responses of the HMG2 gene. Thus, commencing with a2.5 kilo-base pair (kb) fragment 5′ upstream of the HMG2 transcriptioninitiation site, smaller promoter element fragments are generated usingPCR leaving out incrementally larger tracts of sequences from the 5′-endof the, 2.5 kb, fragment. Gene fusions are constructed by ligating the2.3 kb EcoRI/BglII fragment and each of the smaller, PCR-generatedfragments to β-glucuronidase (GUS) reporter genes. The activities of thefusion genes are examined in transgenic plants to determine the locationof the HMG2 promoter.

6.1. Materials and Methods 6.1.1 Plant and Fungal Material

[0146] Tomato (Lycopersicon esculentum cvs. Gardener and Vendor) andtobacco (Nicotiana tobacum cvs. Xanthi and NC95) plants were grown undergreenhouse conditions. Seedlings for transformation experiments weregrown axenically as described below. For elicitor treatments ofsuspension cultured plant cells, tomato EP7 cells were maintained in thedark in a modified MS medium. Verticillium alboatrum (race 1) andFusarium oxysporum (race 1), provided by Dr. Martha Mutschler (CornellUniversity, Ithaca, N.Y.), were maintained on 2.4% potato dextrose agarand grown in 2.4% liquid medium for cell wall isolation. Fungalelicitor, the high molecular weight material heat-released from isolatedmycelial walls was obtained and measured as described (Ayers et al.,1976, Plant Physiol. 57:760-765). Rhizoctonia solani strains RS51 andR992 were provided by Dr. Charles Hagedorn (Virginia PolytechnicInstitute and State University).

6.1.2. Genomic Library Screening

[0147] Recombinant clones (500,000) of a tomato genomic DNA library (L.esculentum cv. VFNT Cherry) constructed in lambda Charon 35 werescreened by plaque hybridization. Hybridization probe was the 1.75 kbEcoRI fragment of pJR326, (provided by Dr. Jasper Rine of University ofCalifornia, Berkeley, Calif.) which contains the region of S. cerevisiaeHMG1 most highly conserved with hamster HMGR (Basson et al., 1986, Proc.Natl. Acad. Sci. USA 83:5563-5567). Initial screening was at lowstringency conditions (e.g., 30% formamide, 6×SSC, 5× Denhardt solution,0.1% SDS, 100 ug/ml salmon sperm DNA at 37° C. for 24 hours; final washconditions were 0.2×SSC at room temperature). Plaques giving positivehybridization signals were carried through at least three rounds ofpurification prior to further characterization. A 7 kb HindIII fragmentof one clone (designated TH29) was subcloned into the HindIII site oftranscription vector pSP6/T7 (Bethesda Research Laboratories (BRL),Gaithersburg, Md.) and designated pTH295 (FIG. 3).

6.1.3. Nucleic Acid Isolation

[0148] Total DNA was isolated from tomato leaves according to the methodof Draper and Scott, (Plant Genetic Transformation and Gene Expression,1988, Eds. Draper et al., Blackwell Scientific, Palo Alto, Calif.,pp211-214)). For RNA isolation, suspension cultured tomato cells treatedwith elicitors, healthy roots, stems, and leaves treated by wounding(cut into 1 mm slices with a razor blade and compressed with a pestle),or intact fruit at various stages of development were stored at −70° C.Total RNA was isolated from 1 to 3 g fresh weight of tissue ground inliquid nitrogen and homogenized directly in a phenol:0.1 M Tris (pH 9.0)emulsion as described previously (Haffner et al., 1978, Can. J. Biochem.56:7229-7233).

6.1.4. Hybridization Analysis

[0149] For genomic Southern analyses, 10 μg/lane total DNA was digestedwith restriction endonucleases, separated on 0.8% agarose gels, andtransferred to Nytran membranes using conditions recommended bymanufacturer (Schleicher and Schuell, Keene, N.H.). For Northernanalyses, total RNA (5 to 20 μg/lane) was denatured by treatment withglyoxal prior to electrophoresis in 1.2% agarose for gel analyses orapplication directly to Nytran filters utilizing a slot blottingapparatus. For hybridizations aimed at revealing all members of the HMGRgene family, probes derived from the 3′ end of the gene which is mosthighly conserved between species (Basson et al., 1988, Mol. Cell. Biol.8:3797-3808) were used. Either the 1.5 kb EcoRI fragment of pTH295 (FIG.3; Yang et al., 1991, Plant Cell 5:397-405) or a 486 bp HMG2 cDNA clonefrom this region derived from pCD1 (NRRL Accession No. ______) were32P-labeled by random-primer methods (Multi-prime Labeling System,Amersham, U.K.). Membranes were prehybridized overnight without labeledprobe and hybridized in the presence of 32P-labeled probe for 24 to 48hr at 42° C. in solution containing 40% formamide, 6×SSC, 5× Denhardtsolution, 5 mM EDTA, 0.1% SDS, 100 μg/ml salmon sperm DNA (Sigma). Finalwash conditions were 0.1×SSC, 0.1% SDS, 1 hr at room temperature. Foranalyses aimed at monitoring hybridization of sequences specific onlyfor the HMG2 isogene encoded by pTH295, the 0.7 kb AvaI-EcoRI fragmentencoding 5-untranslated regions and the 5′ end of the gene or a smaller340 bp subclone derived from this fragment and lacking the upstreamregion, was utilized. Hybridization conditions were as described aboveexcept that 50% formamide and 5×SSC were used. Following hybridization,membranes were washed (final wash used 0.1×SSC at 50° C. for 1 hr) toremove unbound label prior to X-ray film exposure.

6.1.5. HMG2 Promoter:Reporter Gene Fusions

[0150] The 2.5 kb EcoRI fragment of the HMG2-containing clone pTH295 wasinserted into the EcoRI site of a Bluescript SK-vector (Stratagene) anddesignated pDW101 (FIG. 3; NRRL Accession No. ______). Digestion of thisvector with EcoRI and BglII releases a fragment (about 2.3 kb) whichcontains sequences comprising sequences encoding the first fiveN-terminal amino acids of the HMG2 HMGR and extending approximately 2.3kb 5′ upstream of these coding sequences. This fragment was ligated tothe GUS gene at an NcoI site to create an in-frame fusion with the ATGstart codon of GUS and inserted into a modified pRK290 plasmid togenerate pSLJ330.1 (FIG. 8). This plasmid was constructed at theSainsbury Laboratory, John Innes Institute (Norwich, U.K.) incollaboration with Jonathan Jones using transformation vectors providedby Dr. Jones. Subsequent HMG2 promoter constructs were inserted intoplant transformation/expression vectors of the pBI series (ClonTech).The pSLJ and pBI plasmids containing the fusion genes were introduced inAgrobacterium tumefaciens strain LBA4404 (Clontech) by tri-parentalmatings using the helper plasmid pRK2013.

6.1.6. Sequencing of HMG2 Promoter Deletions

[0151] Progressively larger deletions from the 3′ and 5′ ends of the 2.5kb promoter region of HMG2 contained in pDW101 were generated utilizingthe Exonuclease III/Mung Bean Nuclease reaction kit purchased fromStratagene (La Jolla, Calif.). Double-stranded pDW101 was digested withBamHI and SacI (both of which cut in the vector portion of the plasmid),resulting in a linear piece of DNA with a 5′ overhang and a 3′ overhang.After phenol:CHCl₃ extraction to remove the restriction enzymes, the DNAwas precipitated with 100% EtOH and dried. The digested DNA was thentreated with 20 units Exonuclease III/μg DNA to create a segment ofsingle-stranded DNA. Aliquots of the reaction were removed every 45seconds and added to tubes which contained Mung Bean Nuclease buffer.Mung Bean Nuclease was then used to digest the single-stranded portionof the DNA. This process was calculated to remove approximately 300 bpfor every time point. The enzymes were extracted by treatment withlithium chloride and the DNA precipitated by the addition of sodiumacetate and ethanol. This process was followed by a fill-in reactionusing the Klenow fragment to ensure the presence of blunt ends forsubsequent ligation. The resulting plasmids were transformed intoEscherichia coli. Double-stranded DNA sequencing was performed usingSequenase 2.0 (United States Biochemical, Cleveland, Ohio) according toprotocols provided by the manufacturer. Electrophoresis was carried outon 5% HydroLink Long Ranger acrylamide gels (AT Biochem, Malvern, Pa.).

6.1.7. Gus Gene Fusions with HMG2 Promoter Deletions

[0152] Deletions in the HMG2 promoter were created using PCRmethodology. The primers used (designated in FIG. 4), and theapproximate size fragments obtained were: primers #22 and #18, 1000 bp;primers #20 and #18, 460 bp; primers #19 and #18, 170 bp. Primers #19,#20 and #21 generated a flanking HindIII site, primer #18 generated aflanking BamH1 site. The PCR reactions contained 1.5 MM MgCl₂, 200 μMeach dNTP, 2.5 units Taq polymerase, 1× Taq polymerase buffer, 100 ngpDW101, and approximately 40 pmol each primer. Three cycles were used:Cycle 1 (1×): 95° C.-5 min, 72° C.-5 min, 58° C.-2 min, 72° C.-15 min,with the addition of the Taq polymerase after the first 72° C.incubation; Cycle 2 (40×): 95° C.-1 min, 58° C.-2 min, 72° C.-3 min;Cycle 3 (1×): 72° C.-15 min. The DNA fragments generated wereelectrophoresed on a 1.4% agarose gel to verify the sizes.Polyacrylamide gel-purified fragments were digested with the restrictionenzymes HindIII and BamHI. These fragments were then ligated into thebinary vector pBI101 which had been similarly digested (FIG. 7). Theresulting plasmids, pDW201 (NRRL Accession No. ______), pDW202 (NRRLAccession No. ______), and pDW203 (NRRL Accession No. ______) weretransformed into E. coli strain DH5α and their sequences verified. Thebinary plasmids were introduced into Agrobacterium strain LBA4404 usingtri-parental mating.

6.1.8. Transformation of Gene Constructs into Plants

[0153] The HMG2 promoter:reporter gene fusions were transformed intotobacco according to the leaf disk transformation protocol of Horsch etal. (Horsch et al., 1985, Science, 227:1229). Prior to co-cultivationwith appropriately engineered Agrobacterium tumefaciens, leaf disksexcised from axenically grown tobacco seedlings were incubated for 8hours on sterile filter paper overlaying a lawn of cultured tobacco“nurse” cells spread on a feeder plate (modified MS medium containingNitsch vitamins, 100 mg/L myo-inositol, 30 gm/L sucrose, 1 mg/L 2,4-D,0.4 mg/L BAP, 8 gm/L agar). Co-cultivation was accomplished bysubmersing the leaf disks in a suspension of A. tumefaciens at aconcentration of 1×10⁹ cells/ml, followed by vacuum infiltration (3×1min). The leaf disks were then returned to the nurse plates andincubated for 48 hours at 25° C. with indirect light. Disks were thentransferred to selection/regeneration plates (MS salts, Nitsch vitamins,100/L mg myo-inositol, 20 gm/L sucrose, 2 mg/L zeatin, 4 gm agar/L)which contain carbenicillin (500 μg/ml) and an appropriate antibioticfor transformation selection. Plates were returned to the growth chamber(25° C., 18 hr light). Resulting shoots were excised and transferred torooting media (MS salts, Nitsch vitamins, 100 mg/L myo-inositol, 30 g/Lsucrose, 4 gm/L agar, and IAA and kinetin at final concentrations of 10μM and 1 μM, respectively). Rooted plantlets were then transferred tosoil and moved to the greenhouse.

[0154] Transformation of tomato was performed in a similar manner withthe exception of the source of tissue. Cotyledons cut from asepticallygrown tomato seedlings were cut under water using a sterile scalpel into0.5-1.0 cm explants. Explants were then incubated on feeder plates for 8hours prior to transformation.

6.1.9. Examination of GUS Expression in Transgenic Plants

[0155] Histochemical analysis of GUS activity in transgenic planttissues was carried out using standard techniques well known in the art.For routine analyses, plant organs or tissue sections were submerged ina solution of 1 mM X-gluc in 50 mM phosphate buffer (pH 7) and 0.1%Triton X-100. Infiltration of the substrate was facilitated by 3×1minute vacuum infiltration. The amount of vacuum infiltration was variedbased on the thickness and hardiness of the tissue to be tested. Tissuewas incubated 4-12 hours at 37° C. Following development of GUSstaining, leaf tissue was generally treated with ethanol (boil 2 minutesin 95% or incubate overnight in 95%) to remove chlorophyll. GUS activitywas also monitored in cell-free extracts using the fluorescent substrateMUG by standard protocols (Jefferson, 1987, Plant Mol. Biol. Rep.5:387-405). GUS activity was expressed as nmol MU/min/μg protein whereprotein was determined by the method of Bradford (Pierce Coomassie PlusProtein Assay Reagent) using BSA as the standard.

6.1.10 Bacterial Infection Induction of HMG2 Promoter Activity

[0156] A 5 ml culture of Erwinia carotovora ssp. carotovora, strainEC14, causal agent of soft rot, was grown overnight from a single colonyin LB medium at 25° C. The culture was centrifuged and resuspended in 1ml distilled water (OD₆₀₀=3.54-3.82). Leaves, 6-8 inches in length, ofgreenhouse grown transgenic tobacco carrying the HMG2:GUS construct wereexcised at the petiole and placed on water-moistened filter paper in alarge petri dish. The tip of a micropipettor was used to gently woundthe top surface of the leaf while depositing 2 μl of distilled water(mock inoculation) or EC14 suspension. The plates were closed and placedin a plastic container with a tightly-fitting lid (e.g., Tupperware)that had been lined with water-saturated paper towels and incubated at28° C. in the dark. Samples were harvested at 24 hour intervals byexcising a leaf section surrounding the site of inoculation using a holepunch or cork-borer and processed for histochemical analysis asdescribed above.

6.1.11 Fungal Infection Induction of HMG2 Promoter Activity

[0157] Seeds of transgenic tobacco were surface sterilized by soaking in30% bleach, rinsed 4 times with sterile water and germinated inautoclaved potting mix (ProMix BX; Premier Brands, Inc, Stamford, Conn.)in 4×4 inch PlantCons (ICN Biomedicals, Inc, Irvine, Calif.). Fungalinoculations were done by placing a fungal-infested oat seed in contactwith the seedling hypocotyl. Rhizoctonia solani strains RS51 and R992were inoculated as agar plugs into moist, sterilized oat seed and grownfor 8 days at room temperature prior to inoculation on plants. At 24hour intervals, seedlings were removed with care was taken to limitcontact to leaves to minimize wounding of the seeding near the site ofinoculation, and the soil removed by dipping the roots into water. Theentire seedlings, 1-2 inches in length, were then processed forhistochemical GUS determination as described above. Tomato seedlingsgrown on agar ({fraction (1/10)}×MS salts plus 1% agar) were alsoinoculated with a small agar plug of plate-grown Rhizoctonia solani.This method of inoculation was less effective than the soil-grownseedling method because the agar medium supported vigorous fungalgrowth.

6.1.12 Nematode Infestation Induction of HMG2 Promoter Activity

[0158] T1 generation tomato seedlings were grown for 10 days on seedgermination media ({fraction (1/10)}×MS salts, and 10 mg/L myo-inositol,3 gm/L sucrose, 6 gm/L agarose) in 4×4 inch Plantcons. Approximately2000 nematodes (either Melodigyne incognita or M. hapla) were applied tothe top of the media; Plantcons were then placed in a 25° C. growthchamber. Seedlings were harvested at 1, 2, 3, 5, and 7 dayspost-innoculation by slowly pulling seedlings from the agarose, gentlyremoving agarose from roots, and cleanly cutting top of seedling. Rootswere analyzed for GUS expression histochemically.

6.1.13 Post-Harvest Wound Induction of HMG2 Promoter Activity

[0159] Leaves approximately 8 inches in length were removed fromHMG2:GUS and 35S:GUS tobacco plants and were wounded by heavily scoringwith a razor blade. Sections of each leaf were immediately removed,weighed, then frozen in liquid nitrogen and stored at −70° C. Additionalsections of leaf were removed, weighed, and frozen at 24 and 48 hourspost-harvest. Leaf extract was obtained by grinding in MUG extractionbuffer (Jefferson, 1987, Plant Mol. Biol. Rep. 5:387-405) with a mortarand pestle. Protein concentrations were determined by the Bradfordmethod. MUG assays were performed according to the method of Jefferson(Jefferson, 1987, id.); activity was expressed as nmol MU/min//μgprotein.

6.1.14 Field Performance of Transgenic Plants Containing HMG2:GUS GeneFusions

[0160] Seeds of two independent transformants of each of two cultivars(Xanthi and NC-95) showing high levels of wound-inducible HMG2:GUSactivity were used for initial field tests. Seedlings of transgenictobacco carrying a 35S:GUS construct (SJL1911, FIG. 9) were also plantedto use as controls. Transgenic seedlings were grown in the greenhousefor approximately 4 weeks prior to transfer to the field. Test Iinvolved 300 plants (seven genotypes) planted (automated planter) at theVirginia Tech Southern Piedment Agricultural Experiment Station inBlackstone, Va. Test II involved 175 plants (7 genotypes) planted as arandomized block in experimental plots at Virginia Tech, Blacksburg, Va.Leaf material is harvested at various times during the growing season(including just prior to and 2 weeks following routine topping of theplants) to determine field levels of basal HMG2 promoter activity andpost-harvest induction of transgene activity. HMG2:GUS expression inresponse to natural pathogen pressure (including, but not limited to,cyst nematodes, aphids, beetle and hornworm predation, tobacco mosaicvirus) is monitored by histochemical analysis.

6.2 Results 6.2.1 HMG2 Promoter Activity

[0161] In order to delineate regulation of HMG2 expression, 2.3 kb ofHMG2 upstream sequences were fused to the GUS reporter gene in plasmidpSJL330.1 and used for Agrobacterium tumefaciens-mediated planttransformation. More than 20 independent GUS-expressing tobaccotransformants containing 1-4 copies of this construct or a 35S:GUScontrol construct generated by transformation with plasmid pSJL1911(FIG. 9; provided by Dr. Jonathon Jones, John Innes Institute, Norwich,U.K.) were generated. The 35S promoter is a high-level constitutiveplant promoter derived from the cauliflower mosaic virus, (Benfey etal., 1989, EMBO J. 8:2195-2202; Fang et al., 1989, Plant Cell1:141-150).

6.2.2 Tissue Specific Activity of the HMG2 Promoter

[0162] Plants expressing the HMG2:GUS constructs provide a powerful toolfor assessing tissue-specificity of HMG2 expression. Histochemicalanalyses of GUS activity indicated that HMG2 is expressed in unstressedplants in the hypocotyl region of seedlings (region of shoot whichbreaks through soil), in trichomes (plant hairs on leaf surfaceimportant in insect, pathogen-resistance, Gershenzon et al., 1992, Anal.Biochem. 200:130-138), and in pollen (FIG. 10). Expression in thesetissues could be defense-related. Previous reports have shown that somedefense-related genes are elevated in pollen (Kononowicz, 1992, PlantCell 4:513-524). However, significant levels of GUS activity in theprimary root of young tomato seedlings were also observed at sites oflateral root initiation. The significance of this expression iscurrently unknown. It does not appear to be related to cell divisionbecause no GUS expression is found in the zone of cell division in theroot tip. The activity of the HMG2:GUS constructs has also beendetermined in ripening fruit of transgenic tomato plants. Fruit atvarious stages of development were harvest and tested for GUS activityin cell-free extracts or analyzed for tissue specificity usinghistochemical assays. Immature fruit (0.5 to 1.5 cm) showed no HMG2:GUSexpression. However, larger fruit (3-5 cm), mature green fruit (fullsize but without carotenoids), breaker fruit (undergoingcarotenogenesis) and fully ripe fruit showed GUS activity. Histochemicalanalyses identified that the GUS activity was localized primarily to thedeveloping seeds and fruit vasculature. The fruit showed dramaticwound-inducibility of the HMG2:GUS activity. HMG2:GUS activity was alsolocalized to the developing seeds within the seed pods of tobacco.

[0163] Distinct regulatory circuits may mediate inducible versusdevelopmental control of HMG2 expression. During anther (organ producingmale gametophyte) development in immature flowers, wounding induces highlevels of GUS, but immature pollen show no activity. As pollen matures,the pollen expresses GUS but anthers no longer show wound-inducibleactivity.

6.2.3 Defense-Related Activity of the HMG2 Promoter

[0164] Wounding by excision or crushing triggered rapid increases in GUSactivity within 1-12 hours. This response was seen in all tissues testedwith the exception of the mature anthers of tobacco (discussed above).Tissues showing marked wound-induced activation of the HMG2:GUSconstruct include, but are not limited to, roots, hypocotyls, and leavesof tobacco and tomato seedlings; roots, stems, petioles, pedicles, allregions of expanding and mature leaves, developing fruit and pods,mature fruit and pods, and petals of mature tobacco and tomato plants.

[0165] Inoculation of seedlings or excised leaves with compatiblebacterial pathogens triggered specific HMG2:GUS expression localized tothe cells directly surrounding the bacterial lesion. This response wassignificant at 24 hours post-inoculation (the earliest time monitored)as shown in FIG. 11, Panel A. The fungal pathogen, Rhizoctonia solani,also triggered dramatic increases in HMG2:GGS activity (FIG. 11, PanelB) in inoculated seedlings of both tobacco and tomato. Because thesewidely different microbial pathogens both trigger very similar HMG2activation events, it is likely that other bacterial and fungalpathogens will elicit a similar response. At later times duringRhizoctonia infection, the fungus had spread down the vascular system ofthe plants and was associated with significant HMG2:GUS activity inthese tissues. This suggests that the defense-activation properties ofHMG2 will function against vascular pathogens (e.g., wilt-inducingpathogens) as well.

6.2.4 Nematode Activation of the HMG2 Promoter

[0166] Transgenic plants containing the 2.3 kb HMG2 promoter fused toGUS were analyzed for GUS activity following nematode inoculation.Tomato seedlings, germinated axenically on agar medium, were inoculatedby addition of second-stage juveniles of either Meloidogyne incognita orM. hapla, causal agents of root knot disease. The transgenic cultivarswere susceptible to both species. Prior to inoculation and at varioustimes following addition of juveniles, roots were removed,histochemically assayed for GUS activity, and stained for nematodesusing acid fucsin. Uninoculated roots and roots at 24 and 48 hours afterinoculation showed no GUS activity on root tips (the site of nematodepenetration), but GUS activity was evident at the zone of lateral rootinitiation and at the wound site where the stem was removed. At 24 and48 hours post-inoculation, roots containing juveniles were observedwhich showed no indication of GUS activity (FIG. 12, Panel A). By 72 hrspost-inoculation some roots showed low levels of GUS activity in cellsadjacent and proximal (toward the stem) to the juvenile (FIG. 12, PanelB). By five days post-inoculation, high levels of GUS activity wasevident surrounding nematodes that has initiated feeding (characterizedby a change in nematode morphology). By days 5-7, infected root tipsshowed visible swelling. This galled tissue expressed extremely highlevels of GUS (FIG. 12, Panels C, D and E). The responses to M.incognita and M. hapla did not differ significantly. Observation ofmultiple roots and plant samples suggested that the onset of HMG2activation was closely associated with the establishment of feedingbehavior. This suggests that utilization of this promoter to driveexpression of transgene proteins which are toxic or inhibitory tonematode development would significantly impact disease development.Both the temporal and spacial expression pattern are optimal for rapidand highly localized delivery of nematocides or nematostatic agents.

6.2.5 Activation of the HMG2 Promoter by Natural Predators

[0167] Transgenic tobacco plants grown in the field were tested forHMG2:GUS expression in response to natural predation. Leaf sectionssurrounding sites of localized necrosis due to insect or microbialdamage were excised and tested for localized expression of HMG2:GUS.Leaf tissue that had not been damaged showed no GUS activity. However,the region immediately surrounding the lesion showed intense GUSstaining (FIG. 11, Panel C). Transgenic tobacco plants were planted inBlackstone, Va. in fields known to provide high levels of diseasepressure due to the tobacco cyst nematode (Globodera tobacum ssp.solanaceara) in order to assess HMG2 responses to natural nematodeinfection.

6.2.6 Dissection of HMG2 Promoter

[0168] In order to localize elements mediating defense- andtissue-specific expression, efforts were made to generate a series of 5′promoter deletions. In the central region of the HMG2 promoter,delineated in FIG. 5, are several stretches of ATs which seemed toprevent exonuclease digestion through this area. After repeatedunsuccessful attempts at exonuclease strategies, an alternative strategybased on PCR was used to generate deletions of the HMG2 promoter (seeFIG. 7). These truncated promoters were fused to GUS reporter genes inthe plant expression plasmid pBI101 (Clontech) and introduced intotobacco. The promoter region from −981 to +110 confers both wound andmicrobial pathogen responses as well as trichome and pollen expression(Table 4). However, analyses of multiple independently transformedtobacco plants suggest that the levels of transgene expression driven bythis promoter are less than that of promoters that are larger (e.g., to−2.3 kb) or smaller (e.g., −347 bp). This suggests that this region maycontain a “silencer” and that the region upstream of 891 contains anadditional enhancer-type element. All deletions, including the constructcontaining only 58 bp of HMG2 upstream of the transcriptional start sitecontinue to drive transgene expression in the pollen suggesting thatdevelopmental regulation is distinct from that associated with defense.TABLE 4 DISSECTION OF TOMATO HMG2 PROMOTER ELEMENT HMG2:GUS PROMOTER^(a)INDUCED AND TISSUE EXPRESSION^(b) CONSTRUCT REGION (bp) Wound PathogencTrichomes Pollen SLJ 330.1 −2.3 k to +118 ++ +++ ++ ++ DW 203 −891 to+125 + + + ++ DW 202 −347 to +125 ++ ++ ++ ++ DW 201 −58 to +125 −− −−−− +

6.2.7 HMG2 Cis-Regulatory Elements

[0169] Specific cis-regulatory elements within the HMG2 promoter areidentified based on functional analyses and DNA-protein interactions.For example, the region between bases −58 and −347 contains one or moreelements which direct strong wound and pathogen induction. The −58 to−347 fragment is generated by PCR using primer 21 and a primercomplementary to primer 19 (see FIG. 4). Additional 3′ and 5′oligonucleotide primers synthesized based on sequences within thisregion will generate subfragments for analyses of regulatory activity.Functional analyses entail fusion of these fragments to appropriate“minimal” plant promoters fused to a reporter gene such as the minimal35S promoter (discussed in Sections 5.2 and 5.3, above) and introductionand expression of the resulting constructs in plants or plant cells.Regions containing fully-functional HMG2 cis-regulatory elements willconfer HMG2-specific regulation on this minimal promoter as determinedby wound-, pathogen-, pest-, or elicitor-inducibility or generation ofHMG2 sequence-dependent developmental patterns of GUS expression.Specific cis-regulatory elements are further delineated by gel shift orfootprint/DNase sensitivity assays. The −58 to −347 fragment or otherHMG2 promoter fragments, generated by PCR amplification, are incubatedwith nuclear extracts from control and defense-elicited cells. Forexample, nuclei are isolated (Elliot et al., 1985, Plant Cell 1:681-690)from untreated tomato suspension cultured cells and cells 6-8 hoursafter treatment with fungal elicitor or arachidonic acid (Park, 1990,Lycopersican esculentum Mill., Ph.D. Dissertation, Virginia PolytechnicInstitute and State University; Yang et al., 1991, Plant Cell3:397-405). PCR fragments which interact with nuclear components areidentified based on reduced mobility in polyacrylamide gelelectrophoresis or by digestion with DNase I and analysis ofDNase-insensitive areas on sequencing gels. Defense-responsive elementsmay or may not demonstrate differential patterns upon interaction withcontrol versus elicitor-treated extracts. Promoter regions identified ashaving DNA-protein interactions are further tested by creating chimericconstruct with, for example, “minimal” 35S promoters to determineeffects of the cis-regulatory element on heterologous gene expression intransgenic plants or plant cells. This strategy may separate specificHMG2 promoter functions (e.g., wound from pathogen induction, or defensefrom developmental regulation) and delineate additional or novel uses ofHMG2 promoter elements.

6.2.8. Use of HMG2 Promoter for Developing Nematode Resistant Plants

[0170] Another important use of the present invention is the developmentof namatode-resistant plants. Such resistant plants is developed byusing an HMG2 promoter element to control the expression of aheterologous gene which is or whose product is toxic or inhibitory tonematodes. Key advantages of using the HMG2 expression systems for thispurpose are that 1) the heterologous gene product will be limitedprimarily to tissues undergoing disease-related stress, and 2) theheterologous gene will be strongly activated directly in the tissue ofingress, thus delivering a significant “dose” to the nematode.

[0171] The coding region of potato proteinase inhibitor I or II (Johnsonet al., 1989, Proc. Natl. Acad. Sci. USA 86:9871-9875) is excised byappropriate restriction enzymes and inserted into a plant expressionvector downstream of the HMG2 promoter or a chimeric promoter containingactive cis-regulatory element(s) of the HMG2 promoter. Appropriateenzyme sites or linker fragments are used to assure correct positioningof the coding region with the transcriptional and translationalinitiation sites. The plasmid pDW202 (FIG. 7) is digested with SmaI andSstI and gel purified to obtain the vector/HMG2 promoter fragmentseparate from the fragment containing the excised GUS coding region. Theappropriately digested and processed potato proteinase inhibitor I geneis then ligated into the SmaI/SstI site. The resulting plasmid istransformed into E coli strain DH5α and sequenced to assure correctinsertion and orientation prior to introduction into Agrobacteriumstrain LBA4404 by triparental mating. The engineered LBA4404 is thenused for tobacco leaf disk co-cultivation and the transformed tobacco issubsequently regenerated to mature plants.

[0172] Enzymatic or immuno-detection of proteinase inhibitor I intransgenic plants is done by described methods (Johnson et al., 1989,Proc. Natl. Acad. Sci. USA 86:9871-9875). The effect of the introducedHMG2:Proteinase inhibitor constructs on plant:nematode interactions istested in seedlings inoculated, for example, with root knot (Meloidogyneincognita, M. halpa) or cyst (Globodera tobacum) nematodes or by plantseedlings in fields of known disease pressure. Plants are monitored foroverall health, number of feeding female nematodes, number and severityof galls, and nematode egg production.

[0173] In another strategy, HMG2 promoter-driven expression of ananti-sense RNA may be used to block production of plant compoundsrequired by parasitic nematodes. For instance, root knot and cystnematodes are dependent upon plant sterols for growth and reproduction.Thus, sequences from the tomato HMG1 (sterol-specific) isogene (e.g.,regions with the N-terminus specific for HMG1 and not other HMGRisogenes) or the tomato squalene synthetase gene are introduced into theSmaI/SstI digested vector described above such that the complementaryRNA strand is produced. Expression of these gene constructs intransgenic tomato decreases or blocks sterol production in plant cellswhere HMG2 promoter is active and thus reduces or prevents nematodegrowth and development.

7.0. EXAMPLE Post-Harvest Production Using an Expression ConstructComprising the Tomato HMG2 Operably Linked to Gene of Interest

[0174] Post-harvest production of desired gene products in harvestedplant tissues and cultures depends on the ability of the harvestedmaterial to remain competant for induced gene expression. Theexperiments described below demonstrate such an ability in harvestedleaf tissue. Moreover, in the harvested leaf the HMG2 promoter activitysuperior to that of the often used constitutive CaMV 35 promoter.

[0175] The plants used in these experiments are from the HMG2:GUS orCaMV 35S:GUS transformed tobacco plants described in section 6.2.1.above.

7.1. HMG2 Promoter Is Superior to CaMV 35S Promoter in Post-HarvestProduction

[0176] The yield of a specific transgene, β-glucuronidase (GUS), wascompared for transgenic plants containing the HMG2:GUS construct versusthose containing a 35S:GUS construct. The 35S promoter from thecauliflower mosaic virus is widely used in transgenic plant researchbecause it directs high levels of transgene expression in most tissues(Benfey et al., 1989, EMBO J. 8:2195-2202). Leaf material was harvestedfrom greenhouse-grown plants and wounded by scoring with a razor blade.Tissue was extracted for GUS activity immediately after harvest/woundingand after 24 and 48 hours of room temperature storage in zip-lock bagsto prevent desiccation. As shown in FIG. 13, the leaves containing the35S:GUS construct showed greater that 50% loss of GUS activity within 24hours of harvest. For industrial utilization of transgenic tobacco forbioproduction of a high-value protein, this loss would be highlysignificant. In contrast, the HMG2:GUS construct was less activeimmediately after harvest, but by 48 hours, directed wound-inducedtransgene product accumulations to levels comparable to pre-storage35S:GUS levels.

[0177] A transgenic plant carrying a heterologous gene encoding avaluable product, for example a human therapeutic protein ofpharmaceutical value, fused to the HMG2 promoter would be expressed atrelatively low levels throughout the growing cycle of a transgenic plant(e.g., tobacco). Thus, transgene expression is unlikely to affectbiomass yield. Upon optimal biomass production, the plants are harvested(e.g., by mowing) and the leaf material is transferred to the laboratoryor processing facility. HMG2-driven heterologous gene expressiontriggered by mechanical wounding or wounding plus chemical or elicitortreatment and the transgene product is then recovered for furtherprocessing accumulation. The time range of product accumulation isgenerally (but not limited to) 24 to 72 hours depending on the time andconditions between harvest and induction and the stability properties ofthe specific heterologous gene product.

[0178] Heterologous gene products whose production or high-levelaccumulation is deleterious to the growth or vigor of the plant cansimilarly be produced by utilizing the post-harvest inductioncharacteristics of the HMG2 promoter. For highly toxic products it isadvantageous to further dissect the promoter to minimize developmentalexpression but retain the wound and elicitor inducible responses.Promoter deletion analyses (Table 4) indicate that multiplecis-regulatory elements are present in the promoter and that thedevelopmental functions (e.g., pollen) are separable from thedefense-specific functions (e.g., wound, pathogen responses). Theseanalyses indicate that the wound and pathogen response elements aredistal to those elements mediating pollen expression.

7.2. Harvested Plant Tissue Maintain Inducible Gene Expression

[0179] The usefulness of post-harvest production of desired geneproducts depends in part on the ability of the harvested plant tissue orculture to maintain inducible gene expression for some time afterharvest. Such an ability means that the method of invention would haveenhanced industrial utility in as much as it may not always be practicalto induce and process the plant tissue or culture immediately afterharvesting the material. For example, the harvested material may requiretransportation to a distant site for induction or processing.

[0180] Leaves from grown tobacco plants engineered with the HMG2:GUSconstruct were harvested and either induced immediately or after 2 weeksof storage at 4° C. in sealed containers. The results shown in FIG. 14indicate that the stored leaves not only remained competant for theinduced expression of HMG2:GUS construct but actual expressed nearly40-50% more activity than freshly harvested leaves. FIG. 14 also showsthat the induced material requires some incubation time, up to 48 hr.,at room temperature to allow for expression of the induced gene product.

[0181]FIG. 15 shows that the harvested leaves can be stored either at 4°C. in sealed containers or at room temperature in air permeablecontainers for up to 6 weeks and still have ability equal to that of thefreshly harvested material for the expression of inducible genes.

8. DEPOSIT OF MICROORGANISM

[0182] The following microorganisms are deposited with the AgriculturalResearch Culture Collection, Northern Regional Research Center (NRRL),Peoria, Ill. and are assigned the following accession numbers:Microorganism Plasmid Accession No. Escherichia coli DH5α pCD1Escherichia coli DH5α pDW101 Escherichia coli DH5α pDW201 Escherichiacoli DH5α pDW202 Escherichia coli DH5α pDW203

[0183] Although the invention is described in detail with reference tospecific embodiments thereof, it will be understood that variationswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

[0184] Various publications are cited herein, the disclosure of whichare incorporated by reference in their entireties.

1 5 1212 base pairs nucleic acid double unknown cDNA 1 AAAGTCCAGCGCGGCAACCG GGTTCCTCTA TAAATACATT TCCTACATCT TCTCTTCTCC 60 TCACATCCCATCACTCTTCT TTTAACAATT ATACTTGTCA ATCATCAATC CCACAAACAA 120 CACTTTTTCTCTCCTCTTTT TCCTCACCGG CGGCAGACTT ACCGGTGAAA AAATGGACGT 180 TCGCCGGAGATCTGAAGAGC CTGTTTATCC ATCTAAGGTC TTTGCCGCCG ATGAAAAACC 240 TCTCAAACCCCACAAGAAAC AACAACAACA ACAAGAGGAC AAGAATACCC TTCTCATTGA 300 TGCTTCCGATGCTCTCCCAC TTCCTTTGTA TCTCACGAAT GGCTTGTTTT TCACCATGTT 360 TTTCTCTGTTATGTATTTTC TTCTATCAAG GTGGCGTGAG AAAATCAGGA ATTCCACTCC 420 TTTACATGTCGTTACGCTTT CTGAATTGGG TGCTATTGTT TCGTTAATTG CTTCTGTCAT 480 TTATCTTCTTGGTTTCTTTG GGATTGGGTT TGTTCAGACG TTTGTGTCAA GGGGAAATAA 540 TGATTCATGGGATGAAAATG ATGAGGAATT TCTATTGAAG GAAGATAGTC GTTGTGGGCC 600 TGCAACTACTCTTGGTTGTG CTGTTCCTGC ACCACCTGCT CGACAAATTG CCCCAATGGC 660 ACCACCTCAACCTTCTATGT CTATGGTAGA GAAACCTGCA CCGTTGATAA CATCAGCTTC 720 GTCTGGGGAAGACGAAGAGA TAATTAAATC CGTGGTGCAG GGGAAAATAC CATCATACTC 780 ATTGGAATCCAAGCTCGGTG ATTGTAAGCG CGCTGCTTCG ATAAGGAAAG AGGTGATGCA 840 GAGGATTACAGGGAAGTCTC TAGAAGGGCT ACCATTGGAA GGATTTAACT ATGAATCTAT 900 TCTTGGGCAGTGTTGTGAGA TGCCAATTGG GTACGTGCAG ATACCAGTGG GAATAGCAGG 960 GCCATTGTTGCTTAACGGAA AGGAGTTTTC GGTGCCCATG GCAACCACAG AAGGATGTTT 1020 AGTGGCTAGCACCAACAGAG GTTGCAAGGC TATCTATGCT TCTGGTGGTG CTACATGCAT 1080 TTTGCTTCGTGATGGTATGA CCAGAGCACC ATGTGTCAGG TTCGGCACAG CCAAAAGGGC 1140 AGCAGAGTTGAAGTTCTTTG TTGAAGATCC CATAAAATTT GAGTCACTTG CTAACGTTTT 1200 CAACCAGTAAGT 1212 1207 base pairs nucleic acid double unknown cDNA 2 AAATTGTAGCGCGCCAACCG CTTTTCACTC TATAAATACG TTTCTTCTAC TTTCGCTTCC 60 CCACACAAACCATCACTGCT TAATACACAT TTGACTTTCT CTCTCTCTCA TTTCTCTTTT 120 TTTTTTCTTTTCAAAAAGCC GGTGTTCCTG CCGGAAAATC AACTAAATTT ACAATGGACG 180 TTCGCCGGCGACCAGTTAAG CCTTTATGCA CATCAAAAGA TGCTTCCGCC GGCGAACCTC 240 TGAAACAACAACAAGTTTCT AGTCCTAAAG CATCCGACGC GCTTCCACTC CCATTGTACC 300 TAACCAATGGGTTGTTTTTC ACCATGTTTT TCTCTGTTAT GTATTTTCTT CTCGTAAGGT 360 GGCGTGAGAAGATCCGTAAT TCTATTCCTC TTCATGTGGT TACCCTTTCT GAATTGTTAG 420 CTATGGTGTCATTGATTGCT TCCGTTATAT ATCTATTGGG TTTCTTTGGG ATTGGGTTTG 480 TTCAGTCGTTTGTGTCCAGG TCGAATAGTG ATTCATGGGA TATTGAGGAT GAGAATGCTG 540 AGCAGCTAATTATCGAGGAA GATAGCCGCC GGGGACCATG TGCTGCTGCA ACTACTCTTG 600 GCTGCGTTGTGCCTCCACCA CCTGTTCGAA AAATTGCCCC AATGGTTCCA CAGCAACCTG 660 CCAAGGCAGCTTTGTCCCAA ACGGAGAAGC CTGCGCCAAT AATTATGCCA GCATTATCGG 720 AAGATGACGAGGAGATAATA CAATCTGTTG TTCAGGGTAA AACACCATCA TATTCTTTGG 780 AATCAAAGCTTGGTGATTGT ATGAGAGCTG CTTCGATTCG AAAAGAGGCG TTACAGAGGA 840 TCACAGGGAAGTCATTGGAA GGGCTCCCAT TGGAAGGATT TGACTATGAG TCTATTCTTG 900 GACAATGCTGTGAGATGCCT GTAGGATATG TGCAAATACC GGTTGGTATT GCAGGGCCTT 960 TGTTGCTTGATGGGAGAGAG TACTCAGTGC CAATGGCAAC TACAGAAGGG TGTTTAGTTG 1020 CTAGCACCAACAGGGGTTGC AAGGCTATCT TTGTCTCTGG TGGCGCCAAC AGCATTTTGC 1080 TCAGAGATGGCATGACAAGA GCTCCGGTTG TCCGGTTCAC CACCGCCAAA AGAGCCGCAG 1140 AGTTGAAATTCTTCGTTGAG GATCCCCTTA ACTTTGAGAT TCTTTCCCTT ATGTTCAACA 1200 AGTGAGT 12071388 base pairs nucleic acid double unknown DNA (genomic) 3 AAATTGCTGAGATACCTCCT TTGTCTTTTT TCCACTGTGG TCTCAGTTGA CTATAAGAAA 60 TGTTGCTAACTATTTTTAAT GTCCAAACCT CAAATATCAA GCCATGCAAA ATTAACCATT 120 TTCTTAACGTGGATATATAC CTATTCCACC ACATGGTACC CTATTCCAAC TATATTAAAA 180 AAAATAAAATACTCTAGTTA ATATTGATCA AAATTTATAT AATTTACATC TCAATAATAT 240 AAAAAGTTATTGCATGCGTA CTTATGAAAT TTGTAGGTTT TAAAATTGTG TAGGGCTGGG 300 CATAGAAAATTGAATTATCA GATCGGATGG ACAGTTTTTT GGTATTTGGT ATTCGGTAAT 360 TAAGTATTTAATATGATATT TGGAAGTACA ATTTTAGAAT CATAATATTC TGAGTTTGGT 420 ATGAAATATTAGTACCATTT AATATTTTTC GTATACCGAA TTATTTATGA AGGCATGTAT 480 CGACATGTCCATTATTCATT AGTACAAATT TAAAGAGAAA GTTAAAAGAA TAAATATAAA 540 AAATGTAAATACATGTCTTT TAGTTGTATC AATTTATTTT TCTTGATATC TTTTTATTTA 600 TTAATTTTTTTAAATTGTAC CCTGCATAAA AGAAAATAGA AATAATTGGA AAAAAAGTAT 660 TATTTTTATAATACAATCCG ATATAATTAC AATACGATAT TACCGAATAT TATACTAAAT 720 CAAAATTTAATTTATCATAT CAAATTATTA AACTGATATT TCAAATTTTA ATATTTAATA 780 TCTACTTTCAACTATTATTA CCTAATTATC AAATGCAAAA TGTATGAGTT ATTTCATAAT 840 AGCCCAGTTCGTATCCAAAT ATTTTACACT TGACCAGTCA ACTTGACTAT ATAAAACTTT 900 ACTTCAAAAAATTAAAAAAA AAAGAAAGTA TATTATTGTA AAAGATAATA CTCCATTCAA 960 AATATAAAATGAAAAAAGTC CAGCGCGGCA ACCGGGTTCC TCTATAAATA CATTTCCTAC 1020 ATCTTCTCTTCTCCTCACAT CCCATCACTC TTCTTTTAAC AATTATACTT GTCAATCATC 1080 AATCCCACAAACAACACTTT TTCTCTCCTC TTTTTCCTCA CCGGCGGCAG ACTTACCGGT 1140 GAAAAAATGGACGTTCGCCG GAGATCTGAA GAGCCTGTTT ATCCATCTAA GGTCTTTGCC 1200 GCCGATGAAAAACCTCTCAA ACCCCACAAG AAACAACAAC AACAACAAGA GGACAAGAAT 1260 ACCCTTCTCATTGATGCTTC CGATGCTCTC CCACTTCCTT TGTATCTCAC GAATGGCTTG 1320 TTTTTCACCATGTTTTTCTC TGTTATGTAT TTTCTTCTAT CAAGGTGGCG TGAGAAAATC 1380 AGGAATTC1388 480 base pairs nucleic acid double unknown DNA (genomic)modified_base 162 /label= n /note= “n=x=Unknown nucleotide”modified_base 356 /label= n /note= “n=x=Unknown nucleotide” 4 CGAATCAACTAGTTATACTC TCTCACTTAT TTTTGTATTT GTACACCTTT GTCTTACGTG 60 GTGCATTCTGTGTTACTCGA TTGAGGCGCA TGCGAGATAT TTGGATGCCA AGCCTAAGTC 120 AAAAAGGTGCAGAAAATTAC AAAAAAAAAT AAAATTCAGA GNGTAATAGG ACGTTAGTTT 180 ATTTAAAGTGTGTTTTTAAA ATTTCAATTA TAATCTACAA AGATACTTCT GCATTATATA 240 TATATATATATATATATATA TATATATATA TATATATATA TATATATATA TATATATATA 300 TAAAAAGAAGACAGGTACAT TGGATTTTAG CTTGTTTGGT GTCCCAGCAA ACAGCNAAGG 360 AGTAGTATTTTAATTAATAA GTAATCAATT TTGGGTGAGA AATTGCTGAG ATACCTCCTT 420 TGTCTTTTTTTTATATATAT ATATATATAT ATATATATAT ATATATATAT ATATATATTA 480 515 basepairs nucleic acid double unknown DNA (genomic) 5 GAATTCGAAG CAACTTTCCTGACTTGTTTG GTCATGCTTA TAGGTTCACC CAGACTTCGC 60 AGCTCATTTG TAATGGAAGACAACTTTGTG AACATGTCAT GTATAGTTTC TCCTTCCTTC 120 CTTCATTTTG AAGTTCTCATATCGTGAGGT GAGCATGTCA ATCTTGGATT CTTTGACTTG 180 TTCAGTTTCT TCATGTGTAGTCAACAAGCA ATCCCAGATT TCTTTAGCAG ACTCACAGGC 240 TGACACTCTA TTGTACTCATCAGGTCCTAT CCCACAGACC ATAAGAGTTT TACCTTTGAA 300 GCCCTTTTCT ATCTTTTTCCTGTCAGCATC ATCATATTTC TGCTTGGGCT TTGGAACAGT 360 GATGGTCTTT CTCATCTTTACTTCATCATT GGACAAGAGT CACTAGTATA ATATCCCATA 420 ACTCGCTATC TTCAGCCATAATCGTGCATT CTAACTTTCC ACCAACTGTA GAAATGTCCA 480 TTCAAACGAG GAGGTCTATGTGATGACTGA CCTTC 515

What is claimed is:
 1. A method for producing a product encoded by aheterologous gene in a plant cell, comprising a) constructing anexpression vector comprising an inducible promoter element, or afunctional portion thereof, operably linked to a second nucleotidesequence encoding a heterologous gene, wherein said inducible promoterelement or a functional portion of said element controls thetranscription of said heterologous gene sequence; b) engineering a plantcell with said expression vector; c) culturing said engineered plantcell; d) harvesting said engineered plant cell; e) inducing theexpression of said heterologous gene; and f) recovering the expressedheterologous gene product from said engineered plant cell.
 2. A methodfor producing a product encoded by a heterologous gene in harvestedplant tissue, comprising a) constructing an expression vector comprisingan inducible promoter element, or a functional portion thereof, operablylinked to a second nucleotide sequence encoding a heterologous gene,wherein said inducible promoter element or a functional portion of saidelement controls the transcription of said heterologous gene sequence;b) engineering a plant with said expression vector; c) cultivating saidengineered plant; d) harvesting tissue from said engineered plant; e)inducing the expression of said heterologous gene; and f) recovering theexpressed heterologous gene product from said engineered plant.
 3. Themethod of claim 1 or 2, wherein said inducible promoter element is anHMG2 promoter.
 4. The method of claim 3, wherein said inducible promoteris a tomato HMG2 promoter or a tomato HMG2 promoter homolog.
 5. Themethod of claim 3, wherein inducing the expression of said heterologousgene is by wounding or elicitor treatment.
 6. The method of claim 4,wherein inducing the expression of said heterologous gene is by woundingor elicitor treatment.
 7. The method of claim 2, wherein said harvestedtissue is leaf, stem, root, flower, seed or fruit.
 8. The method ofclaim 7, wherein said harvested tissue is leaf.